Multistage Delivery of Active Agents

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(12) United States Patent (io) Patent No.: US 10,143,658 B2 Ferrari et al. (45) Date of Patent: Dec. 4, 2018

(54) MULTISTAGE DELIVERY OF ACTIVE 6,355,270 B1 * 3/2002 Ferrari ...... A61K 9/0097 AGENTS 424/185.1 6,395,302 B1 * 5/2002 Hennink et al...... A61K 9/127 (71) Applicants:Board of Regents of the University of 264/4.1 2003/0059386 Al* 3/2003 Sumian ...... A61K 8/0241 Texas System, Austin, TX (US); The 424/70.1 Ohio State University Research 2003/0114366 Al* 6/2003 Martin ...... A61K 9/0097 Foundation, Columbus, OH (US) 424/489 2005/0178287 Al* 8/2005 Anderson ...... A61K 8/0241 (72) Inventors: Mauro Ferrari, Houston, TX (US); 106/31.03 Ennio Tasciotti, Houston, TX (US); 2008/0280140 Al 11/2008 Ferrari et al. Jason Sakamoto, Houston, TX (US) FOREIGN PATENT DOCUMENTS (73) Assignees: Board of Regents of the University of EP 855179 7/1998 Texas System, Austin, TX (US); The WO WO 2007/120248 10/2007 Ohio State University Research WO WO 2008/054874 5/2008 Foundation, Columbus, OH (US) WO WO 2008054874 A2 * 5/2008 ...... A61K 8/11

(*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Akerman et al., "Nanocrystal targeting in vivo," Proc. Nad. Acad. Sci. USA, Oct. 1, 2002, 99(20):12617-12621. (21) Appl. No.: 14/725,570 Alley et al., "Feasibility of Drug Screening with Panels of Human tumor Cell Lines Using a Microculture Tetrazolium Assay," Cancer (22) Filed: May 29, 2015 Research, Feb. 1, 1988, 48:589-601. Becker et al., "Peptide-polymer bioconjugates: hybrid block copolymers generated via living radical polymerizations from resin- (65) Prior Publication Data supported peptides," Chem. Commun.(Camb), 2003:180-181. US 2016/0051481 Al Feb. 25, 2016 Behrens et al., "Measuring a colloidal particle's interaction with a flat surface under nonequilibrium conditions," Fur. Phys. J. E, 2003, 10:115-121. Bianco et al., "Monoclonal antibodies targeting the epidermal Related U.S. Application Data growth factor receptor," Curr Drug Targets, 2005, 6:275-287. (63) Continuation of application No. 11/836,004, filed on Bradbury, J., "Nanoshell destruction of inoperable tumours," Lancet Aug. 8, 2007, now abandoned. Oneel, Dec. 2003, 4:711. Buriak, J.M., "Organometallic chemistry on silicon and germanium surfaces," Chem. Rev . May 2002, 102(5): 1271-1308. (60) Provisional application No. 60/821,750, filed on Aug. Canham, L. T., `Bioactive silicon structure fabrication through 8, 2006, provisional application No. 60/914,348, filed nanoetching techniques," Advanced Materials, 1995, 7(12):1033- on Apr. 27, 2007. 1037. Charnay et al., "Reduced Symmetry Metallodielectric Nanoparticles: Chemical Synthesis and Plasmonic Properties," J. Phys. Chem. B, (51) Int. Cl. 2003, 107:7327-7333. A61K 9/127 (2006.01) Chen et al. "Soluble ultra-short single-walled carbon nanotubes," J. A61K 9/50 (2006.01) Am. Chem. Soc., 2006, 128:10568-10571. A61K 9/51 (2006.01) Cheng et al. "Nanotechnologies for biomolecular detection and A61K 31/165 (2006.01) medical diagnostics," Curr. Opin. Chem. Biol., 2006, 10:11-19. Chiapponi et al., "Tailored porous silicon microparticles: fabrication A61K 31/704 (2006.01) and properties", Chemphyschem., Apr. 6, 2010, v. 11, No. 5, pp. A61K 31/7088 (2006.01) 1029-1035. (52) U.S. Cl. Ciardiello et al., "A novel approach in the treatment of cancer: CPC ...... A61K 9/50 (2013.01); A61K 9/127 targeting the epidermal growth factor receptor," Clin. Cancer Res., (2013.01); A61K 9/1271 (2013.01); A61K 9/51 Oct. 2001, 7:2958-2670. (2013.01); A61K 31/165 (2013.01); A61K (Continued) 31/704 (2013.01); A61K 31/7088 (2013.01) Primary Examiner Janet L Epps-Smith (58) Field of Classification Search (74) Attorney, Agent, or Firm Winstead PC CPC ...... A61K 31/165; A61K 31/704; A61K (57) ABSTRACT 31/7088; A61K 9/127; A61K 9/1271; A61K 9/50; A61K 9/51; A61K 424/45; Multistage delivery vehicles are disclosed which include a A61K 424/489; A61K 514/34; A61K first stage particle and a second stage particle. The first stage 514/44 particle is a micro or nanoparticle that contains the second USPC ...... 514/34, 44 A; 424/450, 489 stage particle. The second stage particle includes an active See application file for complete search history. agent, such as a therapeutic agent or an imaging agent. The multistage delivery vehicle allows sequential overcoming or (56) References Cited bypassing of biological barriers. The multistage delivery vehicle is administered as a part of a composition that U.S. PATENT DOCUMENTS includes a plurality of the vehicles. Methods of making the multistage delivery vehicles are also provided. 6,099,864 A 8/2000 Morrison et al. 6,107,102 A 8/2000 Ferrari 28 Claims, 58 Drawing Sheets US 10,143,658 B2 Page 2

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(56) References Cited Sullivan et al., "Nanotechnology and tumor imaging: seizing an opportunity," Mol. Imaging, Oct. 2004, 3(4):364-369. OTHER PUBLICATIONS Tasciotti et al., "Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications," Nature Saini et al., "Covalent sidewall functionalization of single wall Nanotechnology, Mar. 2008, 3:151-158. carbon nanotubes," J. Am. Chem. Soc., 2003, 125:3617-3621. Thomas et al. "Delivery of nanogram payloads using magnetic Sakamoto et al., "Anti-Biological Barrier Nanovector Technology porous silicon microcarriers," Lab Chip, 2006, 6:782-787. Torchilin, V P., "Multifunctional nanocarriers," Adv. Drug Deliv. for Cancer Applications," Expert Opin. Drug Deliv., 2007,4(4):359- Rev., 2006, 58:1532-1555. 369. Uziely et al., "Liposomal doxorubicin: antitumor activity and unique Salonen et al., "Mesoporous Silicon in Drug Delivery Applica- toxicities during two complementary phase I studies," J. Clin. tions," Journal of Pharmaceutical Sciences, Feb. 2008, 97(2):632- Oncol., 1995, 13(7):1777-1785. 653. Wang et al., "Surface modification of micromachined silicon fil- Salonen et al., "Mesoporous silicon microparticles for oral drug ters," Journal of Materials Science, 2000, 35:4923-4930. delivery: Loading and release of five model drugs," Journal of Yan et al., "Synthesis and Characterization of Silica-Embedded Iron Controlled Release, 2005, 108:362-374. Oxide Nanoparticles for Magnetic Resonance Imaging," J. Nanosci. Schreiber, F., "Structure and growth of self-assembling monolay- Nanotechnol., 2004, 4(1/2):72-76. ers," Progress in Surface Science, 2000, 65:151-256. Yan et al., "The Embedding of Meta-tetra(Hydroxyphenyl)-Chlorin Shriver-Lake et al.,"Antibody immobilization using heterobifunctional into Silica Nanparticle Platforms for Photodynamic therapy and crosslinkers." Biosens. Bioelectron., 1997, 12(11):1101-1106. Their Singlet Oxygen Production and pH-dependent Optical Prop- So et al., "Self-illuminating quantum dot conjugates for in vivo erties," Photoch Photobiol., 2003, 78(6):587-591. imaging," Nat. Biotechnol., Mar. 2006, 24(3):339-343. Yokokawa et al., "Mechanical properties of aerogel-like thin films Soppimath et al., `Biodegradable polymeric nanoparticles as drug used for MEMS," J. Micromech. Microeng., 2004, 14:681-686. delivery devices," J. Control. Release, 2001, 70: 1-20. Zhang et al., "Proteins and cells on PEG immobilized silicon Starodub et al., "Antibody immobilisation on the metal and silicon surfaces," Biomaterials, 1998, 19:953-60. surfaces. The use of self-assembled layers and specific receptors," Bioelectrochemistry, 2005, 66: 111-115. * cited by examiner M4.90

• j•_qh,_Iff

targeting moieties YT Osecond stage particle PEG chains 0 third stage particle A permeation enhancers first stage particle FIG. 1 Blood Vessel Permeation enhancer

First stage particle

Second stage particle (inside third stage particles) Neovascular endothelium 44 Targeting antibody

Tumor cells

FIG. 2A U.S. Patent Dec. 4, 2018 Sheet 3 of 58 US 10,143,658 B2

CO N 44 d F u- U.S. Patent Dec. 4, 2018 Sheet 4 of 58 US 10,143,658 B2

Time effect on LP/SP Aptes loading A 2000, T

1500 i a— LP Aptes U Carboxyl q-dots 01000_- Z5----_ i -}- LPAptesAmino i q-dots a) 500 ~ f5P Aptes Carboxyl q-dots -_- 0 -- SP Aptes Amino 0 15 30 45 60 q-dots time(minutes)

PEG-FITC-SWNTs i 1000

750 -~- LP APTES 2 -a- LP oxidized 500 l -E- SP APTES -f-SP oxidized a~ I E 250 -

o ------0.01 0.1 1 10 100 Concentration (ng/pl) i FIG. 3 U.S. Patent Dec. 4, 2018 Sheet 5 of 58 US 10,143,658 B2

FIG. 4A

LP oxidized particles

2000 ,

1600

1200 `

C 800

400

0 ------0 15 30 45 60 time (minutes)

------LP APTES particles

2000-

1600

1200

800 -

400

0 0 15 30 45 60 time (minutes) ------

Carboxyl q-dots 'Amino q-dots T PEG-FITC-SWNTs FIG. 4B U.S. Patent Dec. 4, 2018 Sheet 6 of 58 US 10,143,658 B2

FIG. 4C SP o)ddzed particles

200 i i 160 i i i 120 i 80 i i 4o i i 0 0 15 30 45 60 i time (minutes)

'* Carboxyl q-dots Amino q-dots PEG-FITC-SWNTs FIG. 4D U.S. Patent Dec. 4, 2018 Sheet 7 of 58 US 10,143,658 B2

LP oxidized particles tl 50 a 40 a 30 20 10 a~ 0 ------30 00 90 180 360 1200 time (minutes)

Amin© Q-dots ■ PEG-FITC-SWNTs

LP APTES particles

50 0 40

3 30 CL 20 1'0 0 ------'::=Ii 30 60 90 180 360 1200 time (minutes)

El Carboxy Q-dots ■ PEG-FITC-SWNTs FIG. 5 U.S. Patent Dec. 4, 2018 Sheet 8 of 58 US 10,143,658 B2

------CarboxyQuantum Dots

100 - Q f II U 80 C ~ U 60 2 0 40 ff ~f 20 E 0 ,. - 0- 4 - 4- ,4- 0.01 0.1 1 10 100 concentration(pM)

Carboxyq-d'ots —m FITC-SWNTs ------s--4-

B Fluorescence quenching after loading 800 - m Q) 600 - v 400 -

200 - S 0 0 10 20 30 40 50 FITC-SWNTs concentration (pglml)

-+- min FITC SWNTs 5 -;- min FITC SWNTs 15 -•- min FITC SWNTs 30 -- min FITC SWNTs 60 FIG. 6A U.S. Patent Dec. 4, 2018 Sheet 9 of 58 US 10,143,658 B2

r+. LP APTES loading ~r —0 carboxyl Q-dots A amino Q-dots

2000

1500

1000

500

0 0 50 100 150 200 TRIS(mM)

LP o)a'dized loading D 2000 - —f— carboxyl Q-dots — amino Q-dots

1500 -

1000

500

0 0 50 100 150 200 TRIS(m M) U.S. Patent Dec. 4, 2018 Sheet 10 of 58 US 10,143,658 B2

/~ Released FITC-SWNTs

(~400 -~

'300

200 U LL 100 0 load wash time (minutes)

❑ LP Aptes ■ LP oxidized

Released FITC-SWNTs B

60 -

W c 20 h Ir

0 --r ------, 15 45 120 240 time (minutes)

❑ LP Aptes ■ LP oxidized ------FIG. 7 U.S. Patent Dec. 4, 2018 Sheet 11 of 58 US 10,143,658 B2

I:1

Neutral liposomes

Cationic liposomes

FIG. 8A FAGS analysis Key Name Parameter Gate

® XLP APTES + neut lip.008 FL2-H G1 i XLP APTES + poslip.009 FL2-H G1 X -P cm + pos, Iip.006 F:. 24 GI XL' ox rEeu ilg:.007 FL2-H GI L:I nei:t lip.002 FL2-1-i G1 LIP pos iip.003 F-2-1-1 1.31

LP control.001 FL1-H G1

C Liposome loading Excel quantification 2000

1500 as 1000-

500- E 0 LP o)od + pas lip LP oxid + neut lip XLP oxid + pas lip XLP o)dd + neat lip XLP APTES + pos XLP APTES + neut lip lip ------U.S. Patent Dec. 4, 2018 Sheet 13 of 58 US 10,143,658 B2

FINCIVOUR, U.S. Patent Dec. 4, 2018 Sheet 14 of 58 US 10,143,658 B2 U.S. Patent Dec. 4, 2018 Sheet 15 of 58 US 10,143,658 B2

FIG. 10A

Nyj a .):A:? r. X .. •S'£> ~ r r.. }}::7o T4 '~..Gi ;~ t , r..,ti FIG. 1'0B U.S. Patent Dec. 4, 2018 Sheet 16 of 58 US 10,143,658 B2

FIG. 10C

FIG. 10D U.S. Patent Dec. 4, 2018 Sheet 17 of 58 US 10,143,658 B2

FIG. 11A LPISP degradation Number of particles

30000

25000

20000

15000 10000

5000

0 0 6 12 18 24 time (hrs)

—0 Saline LP Saline —i SP Saline

LP/SP degradation Number of particles

30000

25000

20000 e ®.r 15000

10000 5000 —?-----o 0 ------0 6 12 18 24 time {hrs}

—O— CCM LP CCM — --A— — SP CCM' U.S. Patent Dec. 4, 2018 Sheet 18 of 58 US 10,143,658 B2

FIG. 11C LPTSP degradation Volume of

250000

200000

150000

100000

50000

0 0 6 12 18 24 time (hrs)

-0 Saline LP Saline -SP Saline

LPTSP degradation Volume of

250000

200000

150000 '~ v

100000

50000 _

0 ------t?------0 f 12 18 24 time (hrs)

--~- - CCM --a- - LP CCM --A -SP CCM FIG. 11D U.S. Patent Dec. 4, 2018 Sheet 19 of 58 US 10,143,658 B2

Oxidized Silicon Microparticles —+— LP TRIS 0 - --- LP CCM'

50006

J '40000 C O C~ 30000

N U 20000 U c 10000 in 0 0 12 24 36 48 60 72 Time (hours)

Oxidized Silicon Microparticles —.— SP TRIS B ------■ - SP CCM 120000 J 100000

C a 80000 60000 U C 40000

20000 0 0 12 24 36 48 60 72 Time (hours) FIG. 1'2A U.S. Patent Dec. 4, 2018 Sheet 20 of 58 US 10,143,658 B2

------APTES Silicon Microparticles— LP TRIS A ... LP CCM

3' 50000 40000 ro a 30000

0 20000 10000 0 0 4 8 12 16 20 24 28 32 36 40 Time (hours)

APTES Silicon Microparticles —+— SP TRIS ■--- SP CCM' 250000 J 200000 aC V3 150000 a~ 0 100000 U C- 0 50000

0 0 4 8 12 16 20 24 28 32 36 40 Time (hours) FIG. 12B U.S. Patent Dec. 4, 2018 Sheet 21 of 58 US 10,143,658 B2

SP oxidized OQ ♦ \♦ NO \v~\~ \S\?= \ox \v\ A ` \\ \\ \i \`♦ \ i\ \ x \ \4 O\`\a' s~3 \ -~s~ \- \ \\~ ^\fix ~\ \~~~~k~ ♦~ \~ ?a \~p x \\ x\ r \ ♦ ~~~jgw\~`\ \ ♦\ tom`\\\\♦~`~ 1 \\\` \ \ \ \' SP Aptes

\ Al '\(i `Q` t ♦~\~~,\\ \\ \~\ ®\~\ \ \ ♦rte\ `

MON

\ n z ~\`\\`~'3.\~. \ ♦NO

\` \ \ \ \ \♦\ \ \\

C\? $a\M. \.... \ C\\~?~?~\`\ moo. :. \.` \~~ \ \ \\~EllNOW,\If.~~ \;.. LP Aptes \\\\\ '~ \\ \\\\\\ \\\2~a~~\~~a ♦ \\\ \ \ ` \ \\¢El , \f c \ \

f \\ \\ \\\\ \ \ \ k~ ♦ \\ak fix\ \~ .\\~.\ \ t. \ \\\\,,, \ \ ~ ,~~\.?~\ M. ~~'♦\\ FIG 13 U.S. Patent Dec. 4, 2018 Sheet 22 of 58 US 10,143,658 B2

B LDH toxicity Control plate 1,5

f 1.2 0 n `* 0.9

......

0 _ _ --- 0 12 24 36 48 60 72 time(day)

♦•• Control -•*--Cis Platinum 9 Triton 1%o FIG. 1'4A U.S. Patent Dec. 4, 2018 Sheet 23 of 58 US 10,143,658 B2

Experimental LDH toxicity C

0.6

cr 0.4

0.2

0 . 0 12 24 36 48 60 72 time (day)

~--- LP ox 1:1 --s--LP ox 1:5 +► LP ox 1:10 r Experimental LDH toxicity

0.6

m 0.4

i 0.2 0 t6

0 0 12 24 36 48 60 72 time (day)

♦-- SP ox 11:1 -+--SP ox 1:5 SPox1:10 FIG. 14B U.S. Patent Dec. 4, 2018 Sheet 24 of 58 US 10,143,658 B2

HUVEC growth curve

2.5 c 0 2J

1.5

CO 0.5

E 0 ------0 12 24 36 48 60 72 time (hrs)

-•- cells 125 -a- cells 250* cells 500 -+- cells 1000 )K cells 2000 ------Control plate

E c b 1.5

0'•5

...-...... t------0 - t 0 12 24 36 48 60 72 time (hrs)

—0—Control --E-- Cis Platinum ---f-- Triton I% FIG. 15A U.S. Patent Dec. 4, 2018 Sheet 25 of 58 US 10,143,658 B2

------LP oxidized particles

2 E 1.5

1 i .. a

0.5 i E 0 0 12 24 36 48 60 72 time (hrs)

—#— LP bs 1:1 LP bs 1:5 - --*• .. LP bs 1:10

D SP oxidized particles 2 I

0 0 12 24 36 48 60 72 time (hrs)

—+—SPbs1:1 - SP bs 1:5 ...*—SP bs 1:10 FIG. 15B U.S. Patent Dec. 4, 2018 Sheet 26 of 58 US 10,143,658 B2

HUVEC+Saline 24 hrs 1000 1000

800 600 v 500 v LD COO 400 400

kill OR

0 IIIIYYIIIIIIII-1----WrilYlYlY1111411Y1i11Y 0 0 200 400 600 8001000 U zuu 4uu buu MU I uuu FSC-H FSC-H' REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 15961 79.05 79.05 290.53 470.71

C c.:

Size (forward scattering)

FIG. 16A-1 U.S. Patent Dec. 4, 2018 Sheet 27 of 58 US 10,143,658 B2

HUVEC+Saline 1000- 800 ,r/r/ryr~ 600—.- a/~rr cm 400 R 1 200- 0 6-260-4,60- 600 8001000 FSC-H

REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 15004 80.67 80.67 296.59 395.24 Apoptotc C"u"

ame trurwaru scattering)

FIG. 16A-2 U.S. Patent Dec. 4, 2018 Sheet 28 of 58 US 10,143,658 B2

HUVFC+Saline 72 hrs 1000 ->.K 1000 800 800 600 .600 CJ) c.3 y 400 W 400 2001 OP",, 200 0 " 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 12615 83.68 83.68 300.89 485.05

C L

Size (forward scattering)

FIG.. 16A-3 U.S. Patent Dec. 4, 2018 Sheet 29 of 58 US 10,143,658 B2

HUVEC+LP oxidized 24 hrs jjillL illifft EMIR EMI 600 = 600 v v C* 400 y 400 aff 0 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 17024 85.12 85.12 314.68 442.01

Size (forward scattering)

FIG. 16B-1 U.S. Patent Dec. 4, 2018 Sheet 30 of 58 US 10,143,658 B2

HUVEC+LP oxidized 48 hrs MM11 itilrIe 800 800 600. 600 V CO 400 400 200 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H

REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 17473 87.37 87.37 309.15 403.36 Apoptotic

W,-,

tl7 ,ti s Q C* CO Size (forward scattering)

FIG. 168-2 U.S. Patent Dec. 4, 2018 Sheet 31 of 58 US 10,143,658 B2

H'UVECAP oxidized 72 hrs W011 i1111111 800 E•1111, 600 v 600 C3 COD 400 400 04111

I t t I I 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H

REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 ~ 16200 81,00 81.00 289,15 411.50 Apoptotic cells a

Size (forward scattering) FIG. 16B-3 U.S. Patent Dec. 4, 2018 Sheet 32 of 58 US 10,143,658 B2

HUVEC+SP oxidized 24 hrs 1000 1000 800 800 OF 600 600 cooCJ C3 Cn 400 COD 400 200 200 0 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H REGION EVENTS GATED TOTAL X MEAN Y MEAN R1 14234 81.80 81.80 306.77 416.30 Apoptotic cells

0 V CD C

tt7 ~ CQ y Size (forward scattering) FIG. 16C-1 U.S. Patent Dec. 4, 2018 Sheet 33 of 58 US 10,143,658 B2

HUVEC+SP oxidized 48 hrs HOW, MOR

[;Yff I s v 600 C.1 600 COD 400 400 aff aih 0 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 12533 82.48 82.48 292.69 465.59 Apoptotic cells

Cj W Size (forward scattering)

FIG. 16C-2 U.S. Patent Dec. 4, 2018 Sheet 34 of 58 US 10,143,658 B2

HUVEC+SP oxidized 72 hrs 1000 1000- HITE 800-

600 :IF 600 Nil v y 400 y 400 alht 200 ,;.., ~• R1 0 0 0 200 400 600 8001000 0 200 400 600 8001000 FSC-H FSC-H REGION EVENTS % GATED % TOTAL X MEAN Y MEAN R1 , 16920 84.60 84.60 276.66 356.31' Apoptotic Palle '1

size tTorwara scattering)

FIG. 16C-3 U.S. Patent Dec. 4, 2018 Sheet 35 of 58 US 10,143,658 B2

250

200

150 a v 100

0 0 200 400 600 800 1000 FL2-H 200 HUVEC + Saline 160 12 hrs

-DPW120

v 80

100( FL2-H 140 HUVEC + 50µg/ml Cis platinum j 12 hrs 01, ca 80

v 60 40 20

p ~ T n 9nn ann 660 800 100( FL2-H FIG, 17A U.S. Patent Dec. 4, 2018 Sheet 36 of 58 US 10,143,658 B2

300

240

180 a v 120

0 0 200 400 600 800 1000 FL2-H 250

200

.v: 150

C3 100

400 600 800 1000 FL2-H FIG. I7B cuu 200 HUVEC + Saline l 80 48 hrs 160 .cO 60 c 0 v 40 M4

E 20

0 0 0 200 400 600 800 0 200 400 600 800 FL2-H FL2-H 150 VEC HUVEC + Saline 200 hrs 120 72 hrs 160 CO* go- 0 120 0 60- 80

40 30J....

0 0 ...... < ..... 0 200 400 600 800 0 FL2-H FIG. 17C HUVEC + LP Aptes 100- 150 mi 12 hrs 120 M2 80- M2 y Ca 90 60 0 V M4 v 40 M4 60 f ------~ 30 20

0 0 200 400 600 800 0 200 400 600 800 FL2-H FL2-H 200 HUVEC + LP oxidized silicon HUVEC + LP Aptes 150 24 hrs 160 24 hrs 120 y 120 90 - O O Cj 60- MQ v 80 30- 40

0 0 400 600 800 FL2-H FL2-H FIG. 17D I

800

Aptes Aptes

LP LP

hrs

+ +

600

HUVEC HUVEC

400 400

FL2-H FL2-H

------M4------{

200 200

0 0

17E

800

FIG.

600

400

FL2-H FL2-H

200

160

200

=120 HUUEC + LP Aptes 12 hrs

0 200 400 600 800 0 200 400 600 800 FL2-H FL2-H 100 HUUEC + LP oxidized silicon 140 M1 24 hrs 120- 80 12 100. y 60 M3 80 0 M4 r.©.~ 60 v 40 40 20- 20 0 am p 0 0 400 600 800 0 200 400 600 800 FL2-H FL2-H FIG. y7F a 60 50 v 40 30 20 10 0 0 200 400 600 800 FL2-H

150 1c

120 W E"l 90 E a a c a C-" 60 v 4

30 2

0 0 200 400 600 800 200 400 600 R00 FL2-H FL2-H FIG. 17G 200 200 HUVEC + degraded LP oxidized silicon HUVEC + degraded LP Aptes 1 an 48 hrs 48 hrs

0 200 400 600 800 0 200 400 600 800 FL2-H FL2-H 150 100 HUVEC + degraded LP oxidized silicon HUVEC +degraded LP Aptes

0n hRi 72 hrs an nn, 72 hrs

400 600 FL2-H FL2-H FIG. 17H R

800

Aptes

hrs

600

degraded

FL2-H FL2-H

400 400

HUVEC

200

0~ 1~

40 40

80 60 60

100

140 100 120

171

v v

8UU

FIG.

silicon

degraded

hrs

,.0t

600

oxidized

HUVEC

400

FL2-H FL2-H

200

90 60

150

120

v U.S. Patent Dec. 4, 2018 Sheet 44 of 58 US 10,143,658 B2

60 ~+ 5o ; HUVEC + 50pglml Cis platinum cell cycle evaluation

40 ------a \~' i d hrs 12 ® hrs 24

10~\ ~

0, apoptotic G1 S G21M cell cycle phase FIG. 18A U.S. Patent Dec. 4, 2018 Sheet 45 of 58 US 10,143,658 B2 U.S. Patent Dec. 4, 2018 Sheet 46 of 58 US 10,143,658 B2

F 60 HUVEC + SP oxidized silicon cell cycle evaluation

p hrs 12 (a hrs 24 hrs 48 hrs 72 50 ® a

30 0

0 apoptotic G1 S G2/M cell cycle phase ------HUVEC + SP Aptes cell cycle evaluation G 60 50

40 0 30 A v 20

apoptotic G1 S G21M cell cycle phase U.S. Patent Dec. 4, 2018 Sheet 47 of 58 US 10,143,658 B2

r C LL ------g

2000, Q-dots 1000 PEG-FITG-SW NTs

1500 750 c C U I U ~ I 0 1000 500 ~ I C

N I N E 500 1, E 250

0 0L ------0:01 1 100 10000 0.01 0.1 1 10 100 i Concentration(nM) Concentration (ngfuk)

0.01 0.1 1 2

FIG. 20 FIG. 21 A FIG. 21 B a LP oxidized b SP addized.

2000 200

1600 160

1200 120

800 80

4M 40

0 0 0 15 30 45 60 0 15 30 45 60 time (minutes) time(minutes) ------LP APT ES SP APTES ri

2000 200

1600 m 160

1200 120

800

E 400 40

0 0 0 15 30 45 60 0 15 30 45 60 time (minutes) time (minutes) FIG. 21C FIG. 21 D FIG. 21E FIG. 21F LP oxidized SP oxidized D 100 100 , o •Q 80 ❑ Amino Q-dots ■ PEG-FITC-SWNTs 80 Cl Amino q-dots! PEG-FITC-SWNTs 60 60, m n °- w 40 U) 40 m 2p 20 ' rr i_ iii > > • p + . 0.5 1 1.5 3 6 20 0.5 1 1.5 3 6 20 time (hours) time (hours)

LP APTES SP APTES

9 100 100 ❑Carboxyl q-dots ■PEG-FITC-SWNTs ❑ Carboxy Q-dots ■ PEG-FrfC-SWNTs 80 ! 80 o f6 60 - 60 - si a I 40 40 N N ~ 20 w 20 CY ~ 0 --- 0 . A A A 0,5 1 1.5 3 6 20 0.5 1 1.5 3 6 20 time (hours) time(hours)

FIG. 21G FIG. 21'H U.S. Patent Dec. 4, 2018 Sheet 51 of 58 US 10,143,658 B2

N N

Q m -dots

-dots

Unloaded

+Q-dots+SWNTs +Q +SWNTs

Unloaded

+Q-dots+SWNTs

+SWNTs

23A

FIG.

7-1

0 ------Loading Releasing c D --~—Carboxyl-Q-dots 500 PEG-FITC-SWNTs 100 PAN Carboxyl Q-dots °. 400 ~ 80 ~ ~ PEG-FITC-SWNTs 300 Q 60

~,...... T 40 1 100 20 o' 0 30 60 90 0.5 1 1.5 3 6 20 time (min) time (h)

FIG. 23B U.S. Patent Dec. 4, 2018 Sheet 54 of 58 US 10,143,658 B2

NO C'7 tV

W T U.S. Patent Dec. 4, 2018 Sheet 55 of 58 US 10,143,658 B2

N d U Flo LL

M N U.S. Patent Dec. 4, 2018 Sheet 56 of 58 US 10,143,658 B2

FIG. 25 U.S. Patent Dec. 4, 2018 Sheet 57 of 58 US 10,143,658 B2

mrs N E N H V IL

O Q N

:------r ------M p CD CO 0 V N

aseajaj;o % `t m Q-dots loading a

2000

1500

1000

E 500

0 0 15 30 45 60 time (min)

FIG. 27 US 10,143,658 B2 2 MULTISTAGE DELIVERY OF ACTIVE forms, dendrimers, gold nano-shells, semiconductor nano- AGENTS crystals, fullerenes, biologically derived nano-constructs, silicon- and silica-based nanosystems and superparamag- CROSS-REFERENCE TO RELATED netic nanoparticulates are described in the literature.sup.49- APPLICATIONS 5 75.

This application is a continuation of U.S. patent applica- SUMMARY tion Ser. No. 11/836,004, filed on Aug. 8, 2007, which claims the benefit 35 U.S.C. § 119(e) of U.S. Provisional In certain embodiments a composition is provided which Patent Application No. 60/821,750 filed Aug. 8, 2006, and 10 comprises at least one first stage particle that is a micro or U.S. Provisional Patent Application No. 60/914,348 filed nanoparticle and that has (i) a body, (ii) at least one surface, Apr. 27, 2007, the disclosures of which are hereby incor- and (iii) at least one reservoir inside the body, such that the porated herein by reference. reservoir contains at least one second stage particle that comprises at least one active agent. STATEMENT REGARDING FEDERALLY 15 In certain embodiments a method is provided which SPONSORED RESEARCH OR DEVELOPMENT comprises administering to a subject a composition comprising: at least one first stage particle, that is a micro or This invention was made with government support under nanoparticle and that has (i) a body, (ii) at least one surface Grant No. W81XWH-04-2-0035 Project 16 awarded by the and (iii) at least one reservoir inside the body, such that the U.S. Department of Defense and Grant No. SA23-06-017 20 reservoir contains at least one second stage particle that awarded by NASA, and Grant No. NCI 1R21CA1222864- comprises at least one active agent. 01 awarded by the National Institutes of Health. The gov- In still another embodiment a method of making a mul- ernment has certain rights in this invention. tistage delivery system is provided which comprises (A) providing at least one first stage particle, that is a micro or BACKGROUND 25 nanoparticle and that has (i) a body, (ii) at least one surface (iii) at least one reservoir inside the body;(B) providing at Technical Field least one second stage particle and (C) loading the at least The present inventions relate generally to the field of one second stage particle inside the reservoir of the first nanotechnology and, in particular, to compositions utilizing stage particle. These and other embodiments, features and micro and/or nanoparticles for delivery active agents, such 30 advantages will be apparent with reference to the following as therapeutic and imaging agents, and methods of making description and drawings and methods of using such compositions. Description of Related Art BRIEF DESCRIPTION OF THE DRAWINGS The past quarter century's progress in the fundamental understanding of health and disease has not translated into 35 FIG. 1 illustrates a multistage delivery vehicle, in accor- comparable advances in clinical medicine. Inadequacies in dance with an embodiment of the invention. The first stage the ability to administer therapeutic moieties so that they particle contains inside second stage particles. The second will selectively reach desired targets with marginal or no stage particles may comprise at least one active agent, such collateral damage has largely accounted for the discrepancy, as a therapeutic agent or an imaging agent. The first stage see, e.g., Langer, R. Nature 392, 5-10 (1998); and Duncan, 40 particle also contains inside an additional agent, such as a R. Nature Rev. Drug Discov. 2, 347-360 (2003). permeation enhancer or an additional active agent, which Ideally, an active agent, such as a therapeutic or imaging may be an imaging agent or a therapeutic agent. Optionally, agent, should travel through vasculature, reach the intended the second stage particles contain third stage particles. target at full concentration, then act selectively on diseased Targeting moieties, such as antibodies, aptamers or ligands, cells and tissues only, without creating undesired side 45 attached to the surface of the first stage particle, facilitate effects. Unfortunately, even the best current therapies fail to localization at the selected body site. attain this ideal behavior by a wide margin. FIGS. 2A-213 illustrate the principle of operation of a Nano-scale and micro-scale drug delivery systems, also multistage delivery vehicle administered intravascularly, in known as `nanovectors', are promising candidates for pro- accordance with an embodiment. The first stage particle viding solutions to the problem of optimizing therapeutic 50 localizes at the targeted vasculature location. Upon the index for a treatment, i.e. maximizing efficacy, while reduc- localization, the particle releases permeation enhancers that ing health-adverse side effects. Even modest amounts of generate a fenestration in the vasculature. Second stage progress towards this goal have historically engendered particles carry targeting moieties, such as antibodies. The substantial benefits across multiple fields of medicine, with second stage particles may permeate through the fenestra- the translatability from, for example, a subfield of oncology 55 tion and target specific cells that carry surface marker to a field as distant as the treatment of infectious disease antigens, using the antibodies. being granted by the fact that the progresses had a single FIG. 3, Panel A, depicts time dependence of amino and common denominator in the underlying technological plat- carboxy modified quantum dots (q-dots) in APTES modified form. For example, liposomes, the first nanovector therapy "large pore" LP and "small pore" nanoporous silicon first to reach health-care fruition over 10 years ago for treatment 60 stage particle. FIG. 3, Panel B, demonstrates an effect of of Kaposi's sarcoma, have also yielded advances in the second stage PEG-FITC-SWNT nanoparticles concentration treatment of breast and ovarian cancers, as well as fungal on their loading into nanoporous silicon first stage particles. infections. In Panels A and B, Y-axis reads mean fluorescence. Today, many hundreds, if not thousands, of different FIGS. 4A-4D demonstrate time dynamics of second stage nanovector technology platforms have joined liposomes, 65 nanoparticles loading into nanoporous silicon first stage each with different properties, strengths, and weaknesses. particles. 4A for "large pore"(LP) oxidized silicon first stage Various nanovector platforms include polymer-based plat- particles; 4B for LP APTES modified silicon first stage US 10,143,658 B2 3 _►, particles; 4C for "small pore" (SP) oxidized silicon first FIGS.9A -9D demonstrate Scanning Electron Microscopy stage particle; 4D for SP APTES modifies silicon first stage (SEM)images of "large pore"(LP) nanoporous silicon first particles. Y-axis reads mean fluorescence. stage particles. FIG. 5 demonstrates time dynamics of second stage FIGS. 10A-10D demonstrate Scanning Electron Micros- nanoparticles release from LP oxidized nanoporous silicon 5 copy(SEM) images of"small pore"(SP) nanoporous silicon first stage particles (Panel A) and LP APTES modified first stage particles. nanoporous silicon first stage particles (Panel B). In Panel A FIGS. 11A-11D demonstrate degradation of nanoporous and Panel B, Y-axis reads released payload (%). silicon particles measured using Z2 Coulter.RTM. Particle FIG. 6A, Panel A, presents the concentration effect of Counter. Y axis in 11A and 11B reads number of particles. Y axis in 11C and 11D reads volume of particles. loading carboxy modified quantum dots and FITC-conju- FIGS. 12A-B demonstrate degradation of nanoporous gated single wall carbon nanotubes (SWNTs). Y axis in silicon particles measured using Inductive Coupled Panel A reads mean fluorescence (%). FIG. 6A, Panel B, Plasma Atomic Emission Spectrometry. FIG. 12A, Panel demonstrates fluorescence quenching of Fluorescein Isoth- A,for LP oxidized silicon particles; Panel B for SP oxidized iocyanate(FITC) conjugated Single Wall Carbon Nanotubes 15 silicon particles; FIG. 1213, Panel C,for LPAPTES modified (SWNT). silicon particles. Panel D for SP APTES modified silicon FIG. 6B relates to optimization of chemical condition for particles. Y axis in FIGS. 12A-12B reads concentration of loading of second stage particles into nanoporous silicon silicon (ng/mL). particles. Nanoporous silicon particles were mixed with FIG. 13 demonstrates biocompatability of nanoporous second stage nanoparticles(Q -dots in FIG. 613, Panel C and 20 silicon first stage particles by presenting bright field micros- Panel D; PEG-FITC-SWNTs in Panel E and Panel F)in the copy images of selected nanoporous silicon particles and presence of increasing concentration of TRIS. High concen- Human Umbilical Vein Endothelial Cells(HUVEC) cells. In tration of TRIS helped in increasing the amount of Q-dots particular, Panel A demonstrates images for small pore loaded into the first stage silicon particles (Panel C and Panel oxidized silicon nanoparticles; Panel B demonstrates images D). On the contrary, loading efficiency of PEG-FITC- 25 for small pore APTES modified silicon nanoparticles; Panel SWNTs reached its peak at 20 Mm TRIS and then decreased C demonstrates images for large pore oxidized silicon nano- at higher TRIS concentrations, Panel E and Panel F. Y axis particles; Panel D demonstrates images for large pore in Panels B-F reads mean fluorescence. APTES modified silicon nanoparticles. In each Panel A-D, FIG. 7 demonstrates data for loading and release respec- from left to right: first image day 0 (2 hrs after particles tively FITC conjugated with SWNT second stage particles 30 addition), second image day 1; third image day 2; fourth image day 3. into LP nanoporous silicon first stage particles. Panel A FIGS. 14A-B demonstrate biocompatibility of nanop- presents load columns, corresponding to the amount of orous silicon particles by presenting data for Lactate Dehy- FITC-SWNT initially loaded in the nanoporous silicon first drogenase(LDH) toxicity assay on HUVEC cells incubated stage particles after exposure to a FITC-SWNT solution 35 with nanoporous silicon nanoparticles. Y axis in Panels A-F prior to washing. Wash columns in Panel A corresponds to reads absorbance at 490 mu. the amount of FITC-SWNT after washing the first stage FIGS. 15A-B present data for MTT proliferation assay on particles. The actual load of the FITC-SWNT in the first HUVEC cells incubated with nanoporous silicon nanopar- stage particles is the amount of FITC-SWNT retained in the ticles. Y axis in FIG. 15A, Panels A-13 and FIG. 1513, Panels first stage particles after washing, i.e., a difference between 40 C-F reads absorbance at 570 mu. the respective values in the load column and in the wash FIGS. 16A-1-16C-3 present FACS 3D Profiles of column. Panel B shows data for release of FITC-SWNT HUVEC cells incubated with Nanoporous Silicon Particles from the first stage particles over time. The total amount of and analyzed for their size and shape. FITC-SWNT released from the first stage particles over FIGS. 17A-17I present FACS 3D Profiles of HUVEC time, i.e., a sum of all the columns in Panel B, substantially 45 cells incubated with Nanoporous Silicon First Stage Par- matches the difference between respective load and wash ticles and stained with propidium iodide to study cell cycle. columns in Panel A. Y axis in Panels A and B reads amount FIGS. 18A-C present statistical analysis of different of FITC-SWNTs (ng). phases of the cell cycle of cells exposed to nanoporous FIGS. 8A-B present data for lipid based second stage silicon first stage particles. Y axis in FIG. 18A, Panels A-C, particles loading into nanoporous silicon first stage particles. 50 FIG. 1813, Panels D-G, and FIG. 18C, Panels H-K reads % In FIG. 8A, Panels A-C show data for cationic and neutral of total cell population. liposomes loaded into I micron nanoporous silicon first FIG. 19, Panels a and b, demonstrate SEM images of a stage particles. Panel A shows confocal microscopy images porous silicon particle. "Large pore" (LP, FIG. 19a) and of neutral liposomes (left) and cationic liposomes (right). "small pore" (SP, Panel b) particle images showing (from Panels B and C present respectively FACS analysis and 55 left to right) the back side, front side, a cross-section, a Excel quantification of neutral and cationic liposome load- closer view of the pores on the back side and of the pores in ing into I micron nanoporous silicon first stage particles. Y the cross-section. The size and shape of the LP and SP axis in Panel B reads particle number and Y axis in Panel C particles are the same, the size and structure of the pores are reads green fluorescence (logarithmic values). In FIG. 813, significantly different. Panel D shows time dynamics of loading liposomes con- 60 FIG. 20 presents results of flow cytometric and fluores- taining Alexa 555 labeled SiRNA into 3.5 micron nanop- cence microscopy analysis of loading of fluorescently orous oxidized silicon first stage particles. Y axis in Panel D labeled Q-dots and PEG-FITC-SWNTs into nanoporous reads mean fluorescence. Panel E and Panel F present silicon particles. An increase in the amount of nanoparticles fluorescent microscopy images visualizing fluorescence in the loading solution resulted in an increase in the mean associated with liposomes containing Alexa 555 labeled 65 fluorescence of silicon particles (.diamond-solid. LP SiRNA into 3.5 micron nanoporous silicon first stage par- APTES+Carboxyl Q-dots LP oxidized+Amino Q-dots .box- ticles. solid. SP APTES+Carboxyl Q-dots .tangle-solidup. SP oxi- US 10,143,658 B2 5 6 dized+Amino Q-dots) measured by flow cytometry (Panels be attracted into the pores, whereas incompatible charges A and B). Fluorescent microscopy (Panels C and D) con- will partially repel the nanoparticles and thus prevent them firmed that the fluorescence associated to first stage particle to enter the pores. was dimmer when the amount of nanoparticles used was FIG. 26, Panels A-B, present data for liposomes contain- lower. Y axis in Panels A and B reads mean fluorescence. s ing SiRNA loaded into nanoporous silicon first stage par- FIGS. 21A-21H present time dependent loading and ticles. Panel A: Alexa 555 fluorescently labeled siRNAs releasing of second stage particles in nanoporous silicon first were encapsulated into nano-liposomes and loaded into the stage particles. Four different types of nanoporous silicon 1 st stage nano-vectors. The data show that the fluorescence first stage particles (LP oxidized (A), LP APTES (B), SP associated with the porous Silicon carrier increased with the oxidized (C), and SP APTES (D) were loaded with different 10 amount of nanoliposomes.Y axis reads mean fluorescence in second stage nanoparticles (Carboxyl q-dots, .box-solid. Panel A. FIG. 26, Panel B,presents a graph showing relative Amino q-dots, tangle-solidup. PEG-FITC-SWNTs)and their amount of liposomes released from first stage nanoporous fluorescence measured by flow cytometry. Histograms in silicon particles. Y axis reads % of total amount of released 21E-21H represent a percentage of second stage particles 15 liposomes. To test the release of nano-liposome from 1st released from the first stage silicon particles with the pas- stage carriers, the assembled multistage particles were incu- sage of time. bated with 10% fetal bovine serum (pH 7.4) and release of FIG. 22 presents confocal microscopy images demon- nanoliposomes from 1 st stage particles was followed along strating concentrated loading of Q-dots in a highly porous time using fluorimetry. Complete unloading was achieved in region of the back side of silicon particle. Panel a shows 20 about 36 h. confocal microscopy images reconstructed in a series of 3 FIG. 27 demonstrates optimization of physical condition dimensional projections showing a single porous silicon for loading of quantum dots and PEG-FITC-SWNTs. Y axis particle rotated to display different vantage points. Panel b in Panel a and Panel b reads mean fluorescence. Porous shows computer generated 3 dimensional models illustrating silicon particles were desiccated in a desiccator over night the rotation of the particle as shown in Panel a. 25 and then mixed with the loading solution containing the FIGS. 23A-23C illustrate simultaneous loading and second stage nanoparticles. Loading efficiency in dry con- releasing of Q-dots and PEG-FITC-SWNTs second stage dition was not significantly different than loading efficiency particles in nanoporous silicon first stage particles. Flow in a wet environment (p value>0.5). cytometry analysis showing background green and red fluorescence of LP APTES particles (Unloaded; FIG. 23A,Panel 30 DEFINITIONS A and Panel B respectively) and the shifts of the fluorescence signals after the incubation with PEG-FITC-SWNTs Unless otherwise specified "a" or "an" means one or (+SWNTs), Q-dots (+Q-dots) and both (+Q-dots+SWNTs). more. Flow cytometry analysis show that PEG-FITC-SWNTs load Biochemical environment of the target body site" refers 35 rapidly and stabilize, while Q-dots gradually load before to one or more intrinsic physiological conditions at the target reaching a plateau, see FIG. 23B, Panel C. The release site, such as pH, salt conditions, temperature, or the presence profiles of the Q-dots and PEG-FITC-SWNTs are both of target specific moieties, effective to initiate and promote unaltered by the presence of another type of nanoparticle release of the particle content. and are both sustained along time, FIG. 23 B, Panel D. 40 `Biodegradable" refers to a material that may dissolve or Confocal microscopy images show the localization of the degrade in a physiological medium or a biocompatible Q-dots (red) and PEG-FITC-SWNTs (green) in a single polymeric material that may be degraded under physiologi- porous silicon particle. FIG. 23C,Panel E,Panel F and Panel cal conditions by physiological enzymes and/or chemical G show bright field, green and red fluorescence respectively, conditions. while Panel h shows overlay of the 3 channels are shown. 45 "Mucoadhesive" refers to the capability of the particle to Yellow display showed co-localization of green and red adhere to the mucosal layer, which lines the entire surface in fluorescent signals. Y axis in FIG.23 B,Panel D,reads mean the small and large intestine. Adherence is mediated by fluorescence; Y axis in Panel D reads released payload. ligands grafted to the surface of the particles, which bind to FIG. 24, Panels a-m, show fluorescent spectroscopy chemical receptors present in mucin or the surface of the images related to incubating nanoporous silicon particles 50 intestinal epithelial cells. loaded with second stage particles with HUVEC cells for 1 "Targeting moiety" is any factor that may facilitate tar- h at 37 C. In FIG. 24, Panels a-d, the second stage particles geting of a specific site by a particle. For example, the are Q-dots; in Panels e-h, the second stage particles are targeting moiety may be a chemical targeting moiety, a PEG-FITC-SWNTs; in Panels j-m, the second stage par- physical targeting moiety, a geometrical targeting moiety or ticles are Q-dots and PEG-FITC-SWNTs. FIG. 24, Panels d, 55 a combination thereof. The chemical targeting moiety may h and m are bright field images showing the details of be a chemical group or molecule on a surface of the particle; particle morphology. the physical targeting moiety may be a specific physical FIG. 25, Panels A-C, present computer models represent- property of the particle, such as a surface such or hydro- ing the three major physical, chemical and electrostatic phobicity; the geometrical targeting moiety includes a size mechanisms responsible for loading and release of second 6o and a shape of the particle. stage nanoparticles in first stage silicon carrier. (Panel A) "Microparticle" means a particle having a maximum Size Dependency: The size of the pores determines the type characteristic size from 1 micron to 1000 microns, or from of nanoparticles that are preferably loaded into the silicon 1 micron to 100 microns. "Nanoparticle" means a particle particle. (Panel B) Dose Dependency: A larger number of having a maximum characteristic size of less than 1 micron. nanoparticles in the loading solution cause an increase in the 65 `Biocompatible" refers to a material that, when exposed number of particles that are loaded into the pores. (Panel B) to living cells, will support an appropriate cellular activity of Charge Dependency: Compatibly charged nanoparticles will the cells without causing an undesirable effect in the cells US 10,143,658 B2 7 8 such as a change in a living cycle of the cells; a change in In a preferred embodiment, a particle of the last stage is a proliferation rate of the cells and a cytotoxic effect. an active agent formulated as a nanoparticle or alternatively the last stage particle contains the active agent inside, while DETAILED DESCRIPTION a particle of any earlier stage per se may or may not 5 comprise an active agent. In some embodiments, in addition In embodiments of the invention, a composition that to targeting a specific body site, a particle of each stage is includes two or more stages of particles, such that particles designed in such a way that it is capable to perform targeted of a later stage (smaller size particles) are contained in release of its content. In embodiments, the number and type particles of an earlier stage (larger particles), will potentially of stages in the multistage delivery vehicle depends on provide one or more advantages for treating, preventing io several parameters, including administration route and an and/imaging a physiological condition, such as a disease, in intended final target for the active agent. An embodiment of a subject, which may be any animal with a blood system a multistage delivery vehicle is illustrated on FIG. 1. (e.g., the subject may be a warm blooded animal, such as First Stage Particle mammal including human being). Embodiments of such In some embodiments, the particle of the first stage is a multistage composition provide one or more of the follow- 15 micro or nanoparticle. In some embodiments, the first stage ing features or advantages: (1) an active agent, such as a particle has a characteristic size of at least 500 microns or at therapeutic agent or an imaging agent, is preferentially least 1 mm. Such a particle may be configured to contain delivered and/or localized to a particular target site in a body inside at least one micro or nanoparticle, which in turn may of a subject. Preferential delivery and/or localization means contain inside at least one particle of a smaller size. The first that an amount or concentration of the active agent delivered 20 stage particle is any particle that is capable of containing to and/or localized at the target site is higher than an amount inside particles of a smaller size. or concentration of the active agent delivered to and/or In some embodiments, the first stage particle is a top- localized at other sites in the body of the subject; (2) a down fabricated particle, i.e., a particle prepared by top- multistage composition sequentially overcomes multiple down microfabrication or nanofabrication methods, such as biological barriers in a body of the subject; and (3) a 25 photolithography, electron beam lithography, X-ray lithog- multistage composition allows for simultaneous delivery raphy, deep UV lithography or nanoprint lithography. A and localization at the same or different target site of potential advantage of using the top-down fabrication meth- multiple active agents. ods is that such methods provide for a scaled up production Biological Barriers of particles that are uniform in dimensions. Following administration, an active agent, such as a 30 The particle of the first stage may be configured to therapeutic or imaging agent, formulated conventionally or overcome at least one of the following biological barriers: a in a nanovector, encounters a multiplicity of biological hemo-rheology barrier, a Reticulo-Endothelial System bar- barriers that adversely impact the agent's ability to reach an rier, an endothelial barrier, a blood brain barrier, a tumor- intended target at a desired concentration. The biological associated osmotic interstitial pressure barrier, an ionic and barrier may be, for example, an epithelial or endothelial 35 molecular pump barrier, a cell membrane barrier, an enzy- barrier, such as a blood-brain barrier or intestinal lumen matic degradation barrier, a nuclear membrane barrier or a endothelium, that are based on tight junctions, that prevent combination thereof. or limit para-cellular transport of an active agent. Each ofthe The first stage particle may have a body that is defined by endo/epithelial barrier includes a plurality of sequential a size and a shape of the particle and one or more reservoirs subbarriers, such as tight junction barriers, that owe their 40 inside the body. One or more second stage particles may be molecular discrimination to one or more zonula occluden contained inside the reservoir. proteins, and one or more additional biological membranes, The body of the first stage particle comprises any appro- such as vascular endothelial basement membrane or a muso- priate material. Preferably, the material of the body of the cal layer of the intestinal endothelium. Cells of the reticulo- first stage particle is biocompatible. In some embodiments, endothelial system may also act as a biological barrier 45 the body of the first stage particle comprises an oxide against an active agent encapsulated inside nanoparticles, as material such as silicon oxide, aluminum oxide, titanium such cells sequester/uptake the nanoparticles. The biological oxide, or iron oxide; a semiconductor material, such as barrier may be also represented by a cell membrane or a silicon; a polymer or a polymer oxide material; or a ceramic nuclear membrane in a cell that an active agent has to come material. In some embodiments, the body of the first stage through. 50 particle comprises a biodegradable material, such as, for Multistage Delivery Vehicle example, nanoporous silicon. The biodegradable material Since the biological barriers are sequential, overcoming may be such that it degrades when exposed to a physiologi- or bypassing such barriers has to be sequential too. Accord- cal medium such as silicic acid. ingly, a delivery vehicle has been developed that, in embodi- In some embodiments, a material of the body of the first ments, acts in multiple stages. Each stage of the vehicle is 55 stage particle is substantially the same in different regions of defined by a particle having a separate intended function, the body. The shape of the first stage particle may depend on which may be different from an intended function of a the administration route. For example, the shape may be particle of another stage. For example, a particle of one stage configured to maximize the contact between the first stage is designed to target a specific body site, which may be particle and a surface of the target site, such as endothelium different from a site targeted by a particle of another stage, 60 surface for intravascular administration or intestinal epithe- and thus to overcome or bypass a specific biological barrier, lium for oral administration. Accordingly, for oral and which is different from a biological barrier being overcome intravascular administration, the first stage particle may or bypassed by a particle of another stage. A particle of each have a selected non-spherical shape configured to maximize subsequent stage is contained inside a particle of an imme- the contact between the particle and endothelium surface. diately preceding stage. A particle of any particular stage 65 Examples of appropriate shapes include, but are not limited may contain an active agent, such as a therapeutic agent or to, an oblate spheroid or a disc. For pulmonary administra- an imaging agent, intended for use at this particular stage. tion, i.e., administration to lungs ofthe subject, the first stage US 10,143,658 B2 I 10 particles may also have a selected non-spherical shape In some embodiments,for subcutaneous administration, a configured to maximize a contact between the particle and characteristic size of the first stage particle is from 50 one of the epithelial tissues in lungs. microns to 1 mm; or from 100 microns to 1 mm. For pulmonary administration, i.e., an administration One of the functions of the first stage particle, in embodi- route, which involves passing of the particle through lungs 5 ments, is localization at a particular target site. For intra- of a subject, the first stage particle may also have a spicular vascular administration, such target site may be a particular shape, which may facilitate entering of the particle from the vasculature site. For example, in anticancer applications, the lungs into a body tissue, not necessarily through the blood targeted vasculature site may be a tumor vasculature, such as circulation. angiogenesis vasculature, coopted vasculature or renormal- Although top-down fabrication allows manufacturing par- 10 ized vasculature. The localization of the first stage particle at ticles having size in a wide range from 50 mu up to several the targeted site may be facilitated by geometrical factors, millimeters, for certain administration routes particular par- such as the size and the shape of the particle. ticle sizes may be preferred. For example, for intravascular For intravascular administration, the localization at the administration, a maximum characteristic size of the par- targeted site may be also facilitated by one or more recog- 15 ticle, e.g., a diameter for a disc-shaped particle, is preferably nition factors on the surface of the first stage particle. The sufficiently smaller than a radius of the smallest capillary. In recognition factor may be a chemical targeting moiety, such humans, such a radius is about 4 or 5 microns. Accordingly, as a dendrimer, an antibody, an aptamer, which may be a the maximum characteristic size of the particle are, in some thioaptamer, a ligand or a biomolecule that binds a particular embodiments, less than about 3 microns, less than about 2 20 receptor on the targeted site. For oral delivery, the chemical microns or less than about 1 micron. moiety may comprise one or more mucoadhesive ligands, as In embodiments, the maximum characteristic size of the described in Table 1 of U.S. Pat. No. 6,355,270. first stage particle is from 500 mu to 3 microns, or from 700 The selectivity of the targeting may be tuned by changing mu to 2 microns. Yet in some embodiments for intravascular chemical moieties of the surface of the particles. For administration in oncological applications, the maximum 25 example, coopted vasculature is specifically targetted using characteristic size of the first stage particle is such that the antibodies to angiopoietin 2; angiogenic vasculature is rec- first stage particle could localize at the targeted vasculature ognized using antibodies to vascular endothelial growth site without penetrating a fenestration in vascular cancer factor (VEGF), basic fibroblast growth factor (FGFb) or endothelium. For such applications, the maximum charac- endothelial markers such as owP3 integrins, while renormal- teristic size of the first stage particle is greater than about 30 ized vasculature are recognized using carcinoembionic anti- 100 mu, or greater than about 150 mu, or greater than about gen-related yell adhesion molecule 1 (CEACAMI), 200 mu. endothelin-B receptor (ET-B), vascular endothelial growth Yet in some embodiments for intravascular administra- factor inhibitors gravin/AKAP12, a scallofldoing protein for tion, the size of the first stage particle is such so that the protein kinase A and protein kinase C, see, e.g., Robert S. particle may penetrate the fenestration. Accordingly, the 35 Korbel "Antiangiogenic Therapy: A Universal Chemosen- maximum characteristic size in such applications is prefer- sitization Strategy for Cancer?", Science 26 May 2006, vol ably less than about 200 mu, or less than about 150 mu, or 312, no. 5777, 1171-1175. less than about 200 mu. In some embodiments, one may For targeting to non-circulating vasculature cells, the select a size of the first stage particle that is selected to be binding between the first stage particle and the molecular a critical radius of normal non-fenestrated vasculature for 40 marker of the targeted vasculature site should be sufficiently targeting fenestrated vasculature as detailed in PCT patent strong to overcome the drag force exerted by the flowing application No. PCT/US2006/038916 "Methods and Com- blood. This objective may be satisfied by having a relatively positions for Targeting Fenestrated Vasculature" filed Sep. large planar surface area for specific binding and a relatively 27, 2006 to Paolo Decuzzi and Mauro Ferrari. low profile in a capillary's blood flow space, i.e., by having For oral administration, it may be preferable to use the 45 the particle of the selected non-spherical shape, such as an first stage particle that has a maximum characteristic size oblate spheroid or a disc. greater than about 2 microns or greater than about 5 microns The recognition factor may be also a physical recognition or greater than about 10 microns. One advantage of using the moiety, such as a surface charge. The charge may be first stage particle of such size for oral administration is that introduced during the fabrication of the particle by using a such particle may be too large to be endocytosed by intes- 50 chemical treatment such as a specific wash. For example, tinal epithelial cells. The endocytosis by intestinal epithelial immersion of porous silica or oxidized silicon surface into cells has at least two potential disadvantages: 1)the content water may lead to an acquisition of a negative charge on the of the first stage particle may be deactivated as it is pro- surface, see, e.g., Behrens and Grier, J. Chem. Phys. 115 cessed by the endothelial cell before it reaches the desired (14), (2001). P. 6716-6761. The surface charge may be also target; 2) the potential toxicity of particular carrier, e.g. of 55 provided by an additional layer or by chemical chains, such the material of the particle, is of greater concern if it is as polymer chains, on the surface of the particle. For endocytosed than if it is cleared through the gastrointestinal example, polyethylene glycol chains may be a source of a tract. negative charge on the surface. Polyethylene glycol chains In some embodiments, for oral administration, the first may be coated or covalently coupled to the surface as stage particle has a size ranging from 500 microns to several 6o described in P. K. Jal, S. Patel, B. K. Mishra, Talanta 62 centimeters, or from 1 mm to 2 cm. (2004) P1005-1028; S.W. Metzger and M. Natesan, J. Vac. For pulmonary administration, the maximum character- Sci. Technol. A 17(5), (1999) P 2623-2628; and M. Zhang, istic size of the first stage particle is preferably less than T. A. Desai and M. Ferrari, Biomaterials, 19,(1998), p 953. about 20 microns but greater than about 5 microns, if a The positive charge is introduced, for example, by coating targeted site is located in the lungs' air passages. For 65 the surface with a basic polymer, such as polylysine or by targeting a site in alveoli, the maximum characteristic site covalently linking to the surface an amino containing mol- may be less than about 5 microns. ecule, such as 3-aminopropyltriethoxysilane. US 10,143,658 B2 11 12 In some embodiments, modeling methods are applied for Morrison and Mosier on Aug. 8, 2000. In some cases, more selecting geometrical factors, such as a size and a shape, than one exogenous stimulation is used together for acti- and/or surface modification, such as chemical modification vating release. and electrostatic modification, of the particle based on one To increase the localization/targeting efficiency, the first or more properties of the selected target site. Such modeling 5 stage particle may utilize multiple, i.e., more than one methods are disclosed, for example, in 1) U.S. Provisional recognition/localization/targeting factors, which preferably Patent Application No. 60/829,075 "Particles for Cell Tar- do not interfere with each other. For example, the first geting" filed Oct. 11, 2006 to Paolo Decuzzi and Mauro particle may have the selected non-spherical shape as dis- Ferrari; 2) U.S. Provisional Patent Application No. 60/891, cussed above and at the same time carry tumor-targeting 584 "Endocytotic particle" filed Feb. 26, 2007 to Paolo 10 antibodies on its surface. Decuzzi and Mauro Ferrari; 3) Decuzzi, P., Causa, F., In some embodiments, the surface of the first stage Ferrari, M. & Netti, P. A. The effective dispersion of particle may be coated with polymer chains partially or nanovectors within the tumor microvasculature Ann Biomed completely. The polymer chains may be added after the

Eng 34, 633-41 (2006); 4) Decuzzi, P. & Ferrari, M. The 15 fabrication of the intravascular stage particle. The polymer adhesive strength of non-spherical particles mediated by chains may be hydrophilic chains, such as polyethylene specific interactions. Biomaterials 27, 5307-14 (2006); 5) glycol (PEG) chains or synthetic glycocalix chains, which Decuzzi, P. & Ferrari, M. The role of specific and non- may be used for overcoming the uptake of the particle by specific interactions in receptor-mediated endocytosis of cell of the reticulo-endothelial system and, thus, extending nanoparticles. Biomaterials 28, 2915-22(2007); 6)Decuzzi, 20 the blood circulation of the intravascular stage particle. The P., Lee, S., Bhushan, B.& Ferrari, M.Atheoretical model for hydrophilic groups may also serve for enhancement of the margination of particles within blood vessels. Ann solubility of the multistage delivery devices. Biomed Eng 33, 179-90 (2005); Decuzzi, P., Lee, S., A variety of materials may be derivatized with polymer Decuzzi, M. & Ferrari, M. Adhesion of microfabricated chains. For example, when the particle's surface material particles on vascular endothelium: a parametric analysis. 25 comprises a polymer, polymer chains may be attached by Ann Biomed Eng 32, 793-802 (2004). linking carboxylic groups of the polymer and amine or In some embodiments, the first stage particle is configured hydroxyl groups in the polymer chains; if the particle's to release the second stage particles from its reservoir at the surface material comprises metal such as gold, the polymer target site. The release of the second stage particles may be chains may be attached to the surface via thiol chemistry; performed according to a variety of mechanisms, including, 30 when the particle's surface material comprises an oxide, but not limited to, diffusion of the second stage particles such silicon oxide, titanium oxide or aluminum oxide, the through the channels connecting the reservoir and the sur- polymer chains may be attached using silane chemistry. face of the first stage particle and degradation or erosion of In addition to one or more second stage particles, the the body the first stage particle. For such a purpose, the first reservoir of the first stage particle may contain one or more stage particle is configured to have a characteristic release 35 time longer than a characteristic delivery time of the first additional agents. Such additional agent may include an stage particle to its target site when administered to the additional active agent, such as a therapeutic agent or an subject. imaging agent, a targeting agent, one or more penetration In some embodiments, the first stage particle is configured enhancers or any combination thereof. The penetration to perform a release of the second stage particles from its 40 enhancer may include one or more compounds listed in reservoir that is sustained over a period of time longer than Table 1. a characteristic delivery time of the first stage particle to its target site when administered to the subject. In some TABLE 1 embodiments, the first stage particle is configured to release Penetration Enhancers the second stage particles over a time period longer than at 45 least 0.5 hr; or longer than at least 1 hr; or longer than at least Class of Enhancer Specific Examples 2 hr; or longer than at least 8 hr; or longer than at least 15 Bile Salts Glyo-deoxycholate hr; or longer than 30 hr. Tamro-dexoycholate In some embodiments, physical localization and/or tar- Tauro-chenodeoxycholate geted release of the first stage particle is initiated by one or 50 Glyco-chenodexycholate more intrinsic physiological conditions at the target site such Taurocholate as pH, salt concentrations or temperature. In some embodi- Glycocholate Glycoursocholate ments, physical localization and/or targeted release of the Tauroursocholate first stage particle is initiated by exogenous stimulation. Dexoycholate Examples of exogenous stimulations include mechanical 55 Chenodeoxycholate activation, such activation by ultrasound; electromagnetic Cholate Ursocholate activation, such an activation by visible, ultraviolet, near- Non-ionic Surfactants Polyoxyethylene (POE) ethers (e.g., Brij, infrared or infrared light, radiofrequency or X-ray radiation; Texaphor) magnetic radiation. For example, the first stage particle may Alkylphenoxy-POEs (Triton, Igepal, Surfonic) 60 comprise a smart polymer, i.e., a polymer that contracts or Anionic Surfactants sodium dodecyl sulfate expand in a response to a specific stimulus, such as light, dioctyl sodium sulfosuccinate temperature or pH. Smart polymers are described, for Lecithins Lysolecithin example, in "In Situ Activation of Microencapsulated Drugs Medium chain glycerides mono-, di-, or triglycerides of C8, CIO, or (MSC-22866)," NASA Tech Briefs, Vol. 24, No.9 (Septem- C12 fatty acids Medium chain fatty acids sodium caprylate ber 2000), page 64; "Externally Triggered Microcapsules 65 sodium caprate Release Drugs In Situ (MSC-22939), NASA Tech Briefs, sodium laurate (April 2OO2), page 50; and U.S. Pat. No. 6,099,864 issued to US 10,143,658 B2 13 14 TABLE 1-continued size, density, size, shape and /or orientation of nanoporous may be configured for efficient loading of the second stage Penetration Enhancers particles. In some embodiments, pore size in nanoporous first stage Class of Enhancer Specific Examples 5 particles may be, for example,from about 1 rim to about 200 Salicylates sodium salicylate rim; or from about 2 nm to about 100 rim. In some cases, Acylcarnitines decylcarnitine pore size in nanoporous first stage particle may be from laurylcarnitine myristoylcarnitine about 3 to about 10 rim or from about 5 to about 7 rim. Yet Acylcholines Laurylcholine in some cases, pore size in nanoporous first stage particle Palmitoylcholine to may be from about 10 to about 60 mu, or from about 20 to Acyl amino acids N-laurylphenylglycine about 40 rim. N-palmitoylglycine Calcium chelators Ethylenediaminetetraacetic acid (EDTA) In some embodiments, to facilitate loading of the second Peptides PZ-peptide stage particles into pores of the porous or nanoporous Zonula occludens toxin (ZOT) material may be modified chemically and/or electrostatically 15 to make it compatible with chemical and/or electrostatical For intravascular administration, the penetration enhanc- surface properties of the second stage particles. For ers may include a basement membrane penetration enhancer example, for loading negatively charged second stage par- that may act against the basement membrane, and/or one or ticles, it may be preferable to use positively charged pore surface. The positive charge may be achieved, for example, more penetration enhancers, that may act against tight 20 by depositing on pore surface an amino-containing mol- junction proteins (TJPs). An example of the basement mem- ecule, such as 3-aminopropyltriethoxysilane. For loading brane penetration enhancer is metalloproteinase, such as positively charged second stage particles, it may be prefer- Collagenase IV, MMP-2, and MMP-9. An example of the able to use negatively charged pore surface. The negative anti-TJP penetration enhancer is zonula occluden toxin. pore surface charge may be accomplished, for example, by Anti-TJP penetration enhancers and strategies for modulat- 25 oxidizing pore surface with water. ing tight junction permeability are detailed, for example, in In some embodiments, the first stage particle has no Gonzalez-Mariscal, L., Nava, P. and Hernandez, S. J. channels at all. Such particle may comprise, for example, a Membr. Biol., 2005. 207(2): p. 55-68. biodegradable material. For oral administration, the material FIGS. 2A-B illustrate the action of penetration enhancer may be designed to erode in the GI tract. As examples, the upon localization of the first stage particle at the targeted 30 first stage particle may be formed of metals, such as iron, vasculature site, in accordance with an embodiment. The titanium, gold, silver, platinum, copper, and alloys and first stage particle releases the permeation enhancers that oxides thereof. The biodegradable material may be also a generate in the targeted vasculature one or more fenestra- biodegradable polymer, such as polyorthoesters, polyanhy- tions, through which the second stage particles penetrate drides, polyamides, polyalkylcyanoacrylates, polyphospha- into the vasculature. 35 zenes, and polyesters. Exemplary biodegradable polymers In some embodiments, the first stage particle has one or are described, for example, in U.S. Pat. Nos. 4,933,185, more channels fluidically connecting the reservoir with the 4,888,176, and 5,010,167. Specific examples of such bio- surface, that may be in contact with endo or epithelial cells. degradable polymer materials include poly(lactic acid), For intravascular administration, such first stage particle polyglycolic acid, polycaprolactone, polyhydroxybutyrate, 40 poly(N-palmitoyl-trans-4-hydroxy-L-proline ester) and poly may be a micro or nano fabricated particle, such as those (DTH carbonate). detailed in U.S. Patent Application Publication No 2003/ In some embodiments, the body of the first stage particle 0114366 and U.S. Pat. No. 6,107,102, and for oral admin- includes two or more regions configured to contain different istration, such first stage particle may be a micro or fabri- populations of second stage particles. For example, the body cated particle, such as the ones disclosed in U.S. Pat. No. 45 of the first stage particle may have a first region configured 6,355,270. to contain a first population of second stage particles and a In some embodiments, the reservoir and the channels are second region configured to contain a second population of pores in the body of first stage particle. In such case, the first second stage particles. For instance, the first stage particle stage particle may comprise a porous or nanoporous mate- formed of porous nanoporous material may be such that its rial. Preferably, the pores of the porous or nanoporous 5o body has two or more porous regions that differ from each material may be controlled to achieve a desired load of the other. The body of the nanoporous first stage particle may next stage particles and a desired release rate. The nanop- include a first porous region and second porous region that orous material with controllable pore size may be an oxide differ from each other in at least one property such as a pore material, such as silicon oxide, aluminum oxide, titanium density, pore geometry; pore charge, pore surface chemistry, oxide, or iron oxide. Fabrication of nanoporous oxide par- 55 pore orientation or any combination thereof. Such first and ticles, also known as sol gel particles, is detailed, for second regions may be configured respectively to contain a example, in Paik J. A. et. al. J. Mater. Res., Vol. 17, August first and a second population of second stage particles. 2002. The nanoporous material with controllable pore size In some embodiments, the first and second populations of may be also nanoporous silicon. For details of fabrication of second stage particles differ in at least one property such as nanoporous silicon particles, see Cohen M. H. et. al. Bio- 60 size; shape; surface chemical modification; surface charge or medical Microdevices 5:3, 253-259, 2003. Control of pore a combination thereof. density, size, shape and/or orientation may be accomplished In some embodiments, the first and second populations of by changing electrical current and etching time during second stage particles contain the same active agent. Yet, in formation of nanoporous silicon from non-porous silicon. some embodiments, the first and second population of Control of pore density, size, shape and/or orientation may 65 second stage particles contain respectively a first active be also accomplished by varying doping in non-porous agent and a second active agent that are different from each silicon used for formation of nanoporous silicon. Thus, pore other. US 10,143,658 B2 15 16 The first and second populations of second stage particles The composition, size and shape of the second stage may be configured to perform different functions. For particle are not particularly limited. For example, for many example, in some embodiments, the first and second popu- administration routes, the second stage particle may be a lations may be configured to target respectively a first and lipid based particle, such as a liposome, a micelle or lipid second target sites that are different from each other. 5 encapsulated perfluorocarbon emulsion; an ethosome; a car- In some embodiments, the first population are configured bon nanotube, such as single wall carbon nanotube; a to target a particular site in a body of the subject, while the fullerene nanoparticle; a metal nanoparticle, such gold second population is configured to free circulate in the blood nanoshell or triangular silver nanoparticle; a semiconductor system of the subject. nanoparticle, such as quantum dot or boron doped silicon In some embodiments, the first and the second population 10 nanowire; a polymer nanoparticle, such as particles made of target the same target site in a body of the subject but biodegradable polymers and ion doped polyacrylamide par- perform a different function at the body site. For example, ticles; an oxide nanoparticle, such as iron oxide particle, a the first population contains a therapeutic agent to be delivered to the target site, while the second population contains polymer coated iron oxide nanoparticle or a silicon oxide particle; a viral particle, such as an engineered viral particle an imaging agent to be delivered to the target site for 15 imaging or visualizing the target site. or an engineered virus-polymer particle; a polyionic particle, The first and the second regions of the body of the first such as leashed polycations; a ceramic particle, such as silica stage particle may be such that at least one of them is a based ceramic nanoparticles, or a combination thereof. biodegradable region. Preferably, both the first and the In some embodiments, the second stage particle is con- second regions of the body of the first stage particle is 20 figured to target a particular target site in a body of the biodegradable. subject. Such target site may be the same or different from The first and the second regions of the body of the first the target site targeted by the first stage particle. stage particle may have different characteristic time for For example, the surface of the second stage particle may releasing the first and the second population of second stage have one or more antibodies that may conjugate with surface marker antigens of certain types of cells. Thus, the second particles. In some embodiments, both characteristic time for 25 releasing the first population of second stage particles from stage particle may selectively target cells that carry such the first region and characteristic time for releasing the marker antigens. The examples of cells that carry surface second population of second stage particles from the second marker antigens include stem or clonogenic cells and tumor region may be greater that a characteristic for delivery cells. The surface marker antigens on stem or clonogenic cells may be targeted by CD33 antibody. A number of and/or localization of the first stage particle at its target site 30 when administered to the subject. monoclonal antibodies to tumor specific antigens are avail- In some embodiments, the first stage particle is configured able, see, e.g., pp. 301-323 of CANCER, 3rd Ed., De Vita, to separate into a first component that includes the first et. al. eds; 7aneway et. al. Immunology 5th Edition, Garland region and a second component that includes the second Press, New York, 2001; A. N. Nagappa, D. Mukherjee & K. Anusha "Therapeutic Monoclonal Antibodies", PharmaBiz. region when being exposed to a physiological medium, such 35 as a medium that may be present at a target site of the first com, Wednesday, Sep. 22, 2004. Table 2 presents FDA stage particle. Such exposure may occur, for example, when approved monoclonal antibodies for treatment of cancer. the particle is administered to the subject. In some embodiments, the first and the second regions of 11OHNIM the body of the first stage particle are configured to perform 40 Trade a different function when the particle is administered to the MAb Name Name Used to Treat: Approved in subject. For example, the first and the second region of the body of the first stage particle may be configured to over- Rituximab Rituxan Non-Hodgkin lymphoma 1997 Trastuzumab Herceptin Breast cancer 1998 come respectively the first and the second biological barriers Gemtuzumab Mylotarg Acute myelogenous 2000 that are different from each other. Such biological barriers 45 ozogamicin* leukemia (AML) may be each selected, for example, from a hemo-rheology Alemtuzumab Campath Chronic lymphocytic 2001 barrier, a Reticulo-Endothelial System barrier, an endothe- leukemia (CLL) Ibritumomab tiuxetan* Zevalin Non-Hodgkin lymphoma 2002 lial barrier, a blood brain barrier, a tumor-associated osmotic Tositumomab* Bexxar Non-Hodgkin lymphoma 2003 interstitial pressure barrier, an ionic and molecular pump Cetuximab Erbitux Colorectal cancer 2004 barrier, a cell membrane barrier, an enzymatic degradation 50 Head & neck cancers 2006 barrier, a nuclear membrane barrier or a combination Bevacizumab Avastin Colorectal cancer 2004 thereof. Second Stage Particle In some embodiments, the second stage particle is con- The second stage particle may be any micro or nanopar- figured to freely circulate in a blood system of the subject ticle that may fit inside the reservoir of the first stage 55 upon being released from the first stage particle. In some particle. For example, in certain embodiments, for oral or cases, such second stage particle may have a surface free of pulmonary administration, the second stage particle is the targeting moieties, such as antibodies. The free circulating same as the first stage particle for intravascular administra- second stage particle may be used, for example, as to report tion. of therapeutic action a therapeutic agent associated with a The particle of the second stage may be configured to 60 first stage particle per se. overcome at least one barrier selected from a hemo-rheology In some embodiments, the surface of the second stage barrier, a Reticulo-Endothelial System barrier, an endothe- particle does not have hydrophilic polymer chains, such as lial barrier, a blood brain barrier, a tumor-associated osmotic polyethylene glycol (PEG) disposed on it. In certain cases, interstitial pressure barrier, an ionic and molecular pump this may be an advantage of the multistage delivery vehicle barrier, a cell membrane barrier, an enzymatic degradation 65 as PEG chains are usually attached to liposomes and other barrier, a nuclear membrane barrier or a combination nanoparticles to delay recognition and sequestration by thereof. macrophages of the reticulo-endothelial system. Unfortu- US 10,143,658 B2 17 18 nately, the PEG chains may also hide antibodies on the may serve for delivering to the cell's nucleus an active nanoparticles surfaces and thus inhibit the targeting/local- agent, that may be an agent acting against nucleic acids or ization ability of the antibodies. In the multistage delivery a gene therapeutic agent. To be able to cross the nuclear vehicle, PEGS attached to the first stage particle may per- membrane, the third stage particle may range from about 3 form shielding from the RES macrophages. Although the 5 mu to about 10 mu. The ability to deliver nanoparticles in 3 PEGS may hide antibodies on the first stage particle, the mu to 10 mu size range may be one of the advantages of the recognition/localization capability of the first stage particle multistage delivery vehicle as a conventional administration is not limited to the antibodies, as other factors such as the of such particles via injection usually results in their imme- particle's size and shape also may contribute to such capa- diate globular clearance. bility. In some embodiments, the surface of the second stage io In some embodiments, the multistage vehicle includes a particle has hydrophilic polymer chains, such as PEG third stage. The third stage may be any nanoparticle that may chains, disposed on it. fit inside the second stage particle. As with the second stage In some embodiments, the surface of second stage particle is modified, for example, to facilitate the second stage particle, the third stage particle may be a lipid based particle, such as a liposome, a micelle or lipid encapsulated perfluo- particle's ability to load into a reservoir of the first stage 15 particle and/or to facilitate the second stage particle's ability rocarbon emulsion; an ethosome; a carbon nanotube, such as to reach its target site. The surface modification may include single wall carbon nanotube; a fullerene nanoparticle; a a chemical modification of the surface of the second stage metal nanoparticle, such gold nanoshell or triangular silver particle and/or electrostatic modification of the surface of nanoparticle; a semiconductor nanoparticle, such as quan- the second stage particle. For example, to facilitate loading 20 tum dot or boron doped silicon nanowire; a polymer nano- of the second stage particles into a porous or nanoporous particle, such as particles made of biodegradable polymers first stage particle, the surface of the second stage particles and ion doped polyacrylamide particles; an oxide nanopar- may be modified so that its properties are compatible with ticle, such as iron oxide particle, a polymer coated iron oxide surface properties of pores of the porous or nanoporous first nanoparticle or a silicon oxide particle; a viral particle, such stage particle. For example, when the pores of the porous or 25 as an engineered viral particle or an engineered virus- nanoporous first stage particle are positively charged it may polymer particle; a polyionic particle, such as leashed poly- be preferably to modify the surface of the second stage cations; a ceramic particle, such as silica based ceramic particles so that they are electrostatically neutral or have a nanoparticles, or a combination thereof. In some embodi- negative surface charge; while when the pores of the porous ments, the third stage particle is a nucleic acid nanoparticle, or nanoporous first stage particle are negatively charged, it 30 such as a small interfering RNA (siRNA) particle. may be preferably to modify the surface of the second stage Loading Later Stage Particles into a Reservoir of Earlier particles so that they are electrostatically neutral or have a Stage Particle positive surface charge. The chemical and/or electrostatic Later stage particles may be introduced into a earlier stage surface modification of the second stage particles may be particle of an earlier stage by any appropriate technique. In performed using the same methods as detailed above to the 35 some embodiments, one may soak nanoporous earlier stage first stage particles. particles fabricated in a solution containing a carrying fluid For lipid containing second stage particles, such as lipo- and the later stage particles, which may enter pores of the somes or micelles, the electrostatic modification may be earlier stage particle via capillary action and/or diffusion. performed by incorporating in their lipid layers lipids that The carrying fluid may be a liquid that is biologically may affect the electrostatic charge of the liposome. For 40 non-harmful and that is neutral with respect to the earlier example, to form a positively charged cationic lipid con- stage particle's material. An example of the carrying fluid taining particle, one may use cationic lipids, such as 1,2- may be phosphate buffer saline (PBS) or a deionized water. Dioleyl-3-trymethylammoniumpropane(DOTAP); to form a The solution may also contain one or more additional agents, negatively charged anionic lipid containing particle, anionic such one or more additional therapeutic agents and one or lipids, such as dioleoylphosphatidyl glycerol (DOPG); and 45 more appropriate penetration enhancers, desired to be intro- to form a neutral lipid containing particle one may use, duced in the first stage particle. To maximize a load of the neutral lipids, such as DOPC. later stage particles, one may use a solution that has a In some embodiments, upon binding to the targeted cell or saturated concentration of the later stage particles. cells, the second stage particle may release its content into The earlier stage particles may be introduced into the the cell's cytoplasm. In some embodiments, such release is 50 solution in a form of suspension. Preparation of nanoporous activated by exogenous factors, such as electromagnetic particles suspension is detailed, for example, in U.S. patent radiation. For fullerene nanoparticles and carbon nanotubes, application Ser. No. 2003/0114366. Pores of the nanoporous such radiation may be radio-frequency radiation, while for particles may be dried prior their submerging in the solution gold-shell nanoparticles, the radiation may be a near-infra- containing the later stage particles. red radiation. Activation of nanoparticulates by exogenous 55 In some embodiments, the solution containing the later radiation is detailed, for example, Hirsch, L. R., Halas, N. J. stage particles pores may be degassed prior to the introduc- & West, J. L. Anal. Chem. 75, 2377-2381 (2003); Hirsch, L. tion of the earlier stage particles. Then, the earlier stage R., Halas, N. J. & West, J. L. Proc. Natl Acad. Sci. USA 100, particles may be submerged in the degassed solution in a 13549-13554(2003); and O'Neal, D.P., Halas, N. J. & West, sealed chamber. The earlier stage particles may be subjected J. L. Cancer Lett. 209, 171-176 (2004). 60 to reduced pressure to ensure that trapped air is forced from In some embodiments, the content of the second stage the pores in the particles. Then the earlier stage particles may particle is one or more active agents per se. In some be fully immersed in the solution and the pressure in the embodiments, the second stage particle contains inside a sealed chamber may be elevated slightly above atmospheric third stage particle, which itself contains inside one or more to make sure that the solution enters the pores of the earlier active agents. The third stage particle may be, for example, 65 stage particles. The earlier stage particles may then be a particle, that is small enough to be able to cross a nuclear trapped on a filter and dried using one of the three methods membrane of the targeted cell. Thus, the third stage particle described below. US 10,143,658 B2 19 20 In some embodiments, to remove any trapped air within surface of the nanoporous earlier stage particles and a the reservoirs in the submerged earlier stage particles, the surface of the later stage particles are modified with chemi- pressure within the chamber is reduced and then raised cal groups compatible with each other. For example, one of slightly above atmospheric pressure. the pore surface of the nanoporous earlier stage particles and In some embodiments, after filling the solution into the 5 the surface of the later stage particles may be modified with pores of the earlier stage particles, drying is achieved by one carboxy groups; while the other may be modified with or more of the following three methods. Water may be amino groups. removed by evaporation under reduced pressure in a vacuum Active Agent chamber, or by passage of a stream of warm air or an inert The active agent may be a therapeutic agent, an imaging gas such as nitrogen over the surface particles collected on io agent or a combination thereof. The active agent may be any a filter, or by freeze drying. In the case of freeze drying, a appropriate agent that may be released from a particle flat heat exchanger may be placed in good thermal contact, containing it. The selection of the active agent depends on e.g. directly below, the filter, on which the earlier stage the application. particles have been collected. Refrigerant fluid at tempera- When the active agent is not a particle of any stage per se, tures ranging from —20° C. to —60° C., such as Freon, or a 15 it may be introduced into particle using any appropriate cold liquid, such as liquid nitrogen, may be passed through technique. For example, when the active agent is doxorubi- the heat exchanger flowing into port and passing out port in cin, it may be introduced in a liposome particle using a order to freeze any water remaining within the pores. The protocol detailed in Working Example 4. pressure may be then reduced until all the water sublimes. When the active agent is a particle of one of the stages of In some embodiments, for loading later stage particles 20 multistage delivery vehicle, the active agent may introduced into nanoporous earlier stage particles, a solution containing using one of the methods disclosed above. the earlier stage nanoporous particles, the later stage par- Therapeutic Agent ticles and a carrier liquid is prepared. The carrier liquid may The therapeutic agent may be any physiologically or be a physiological buffer, such as Tris-HCI. A concentration pharmacologically active substance that may produce a of the carrier liquid may be selected by using standard 25 desired biological effect in a targeted site in an animal, such techniques to maximize loading of the later stage particle as a mammal or a human. The therapeutic agent may be any into the nanoporous earlier stage particles. For example, in inorganic or organic compound, without limitation, includ- some embodiments, for Tris-HCI, the optimal concentration ing peptides, proteins, nucleic acids, and small molecules, may be selected from 1 to 500 mM. any of which may be characterized or uncharacterized. The Geometrical properties of the later stage particles, such as 30 therapeutic agent may be in various forms, such as an size and shape, may be selected to be compatible pore unchanged molecules, molecular complex, pharmacologi- properties of the nanoporous earlier stage particles, such as cally acceptable salt, such as hydrochloride, hydrobromide, pore density, pore size, and pore orientation. sulfate, laurate, palmitate, phosphate, nitrite, nitrate, borate, In some embodiments, loading of the later stage particle acetate, maleate, tartrate, oleate, salicylate, and the like. For into the nanoporous earlier stage particles is facilitated by 35 acidic therapeutic agent, salts of metals, amines or organic agitating the solution containing both the later stage and cations, for example, quaternary ammonium, may be used. earlier stage particles. Such agitation may be performed by Derivatives of drugs, such as bases, esters and amides also spinning the solution in a rotating wheel. Agitation condi- may be used as a therapeutic agent. A therapeutic agent that tions, such as a rotation speed of the rotating wheel, may be is water insoluble may be used in a form that is a water optimized using standard techniques to achieve a desired 40 soluble derivative thereof, or as a base derivative thereof, loading degree and/or loading time. which in either instance, or by its delivery, is converted by In some embodiments, loading of the later stage particles enzymes, hydrolyzed by the body pH, or by other metabolic into the earlier stage nanoporous particles is controlled by processes to the original therapeutically active form. varying a concentration of the later stage particles in the The therapeutic agent may be a chemotherapeutic agent, solution. In some embodiments, the higher load may be 45 an immunosuppressive agent, a cytokine, a cytotoxic agent, achieved by using a higher concentration of the later stage a nucleolytic compound, a radioactive isotope, a receptor, particles in the solution. Yet, in some embodiments, one may and a pro-drug activating enzyme, which may be naturally achieve a higher load of the later stage particles by using a occurring or produced by synthetic or recombinant methods, concentration of the later stage particles, which is lower than or any combination thereof. the highest possible concentration of the later stage particles 50 Drugs that are affected by classical multidrug resistance, in the solution. Determining a concentration maximizing such as vinca alkaloids (e.g., vinblastine and vincristine), the loading of the later stage particles in the nanoporous earlier anthracyclines (e.g., doxorubicin and daunorubicin), RNA stage particles may be performed using standard methods. transcription inhibitors (e.g., actinomycin-D) and microtu- In some embodiments, loading of the later stage particles bule stabilizing drugs (e.g., paclitaxel) may have particular into the earlier stage nanoporous particles is controlled by 55 utility as the therapeutic agent. modifying a pore surface of the nanoporous earlier stage A cancer chemotherapy agents may be a preferred thera- particles and/or a surface of the later stage particles in order peutic agent. Useful cancer chemotherapy drugs include to make them more compatible. Such modifying may be nitrogen mustards, nitrosorueas, ethyleneimine, alkane sul- performed by modifying surface chemical groups on either fonates, tetrazine, platinum compounds, pyrimidine analogs, surface and/or by modifying an electrical charge on either 60 purine analogs, antimetabolites, folate analogs, anthracy- surface. For example, in some embodiments, to achieve a clines, taxanes, vinca alkaloids, topoisomerase inhibitors higher load of the later stage particle, one may use nega- and hormonal agents. Exemplary chemotherapy drugs are tively charged surface of the later stage particles and posi- Actinomycin-D, Alkeran, Ara-C, Anastrozole, Asparagi- tively charged porous surface of the earlier stage nanoporous nase, BiCNU, Bicalutamide, Bleomycin, Busulfan, Capecit- particles or positively charged surface of the later stage 65 abine, Carboplatin, Carboplatinum, Carmustine, CCNU, particles and negatively charged porous surface ofthe earlier Chlorambucil, Cisplatin, Cladribine, CPT-11, Cyclophosph- stage nanoporous particles. In some embodiments, a pore amide, Cytarabine, Cytosine arabinoside, Cytoxan, Dacar- US 10,143,658 B2 21 22 bazine, Dactinomycin, Daunorubicin, Dexrazoxane, Doc- romethylornithine (DMFO); retinoic acid; Esperamicins; etaxel, Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Capecitabine; and pharmaceutically acceptable salts, acids Etoposide, Floxuridine, Fludarabine, Fluorouracil, Fluta- or derivatives of any of the above. Also included are mide, Fotemustine, Gemcitabine, Herceptin, Hexamethyl- anti-hormonal agents that act to regulate or inhibit hormone amine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, 5 action on tumors such as anti-estrogens including for Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, example Tamoxifen, Raloxifene, aromatase inhibiting 4(5)- Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Oxali- imidazoles, 4 Hydroxytamoxifen, Trioxifene, Keoxifene, platin, Paclitaxel, Pamidronate, Pentostatin, Plicamycin, Onapristone, And Toremifene (Fareston); and anti-andro- Procarbazine, Rituximab, Steroids, Streptozocin, STI-571, gens such as Flutamide, Nilutamide, Bicalutamide, Leupro- Streptozocin, Tamoxifen, Temozolomide, Teniposide, Tetra- io lide, and Goserelin; and pharmaceutically acceptable salts, zinc, Thioguanine, Thiotepa, Tomudex, Topotecan, Treosul- acids or derivatives of any of the above. phan, Trimetrexate, Vinblastine, Vincristine, Vindesine, Cytokines may be also used as the therapeutic agent. Vinorelbine, VP-16, and Xeloda. Examples of such cytokines are lymphokines, monokines, Useful cancer chemotherapy drugs also include alkylating and traditional polypeptide hormones. Included among the agents, such as Thiotepa and cyclosphosphamide; alkyl 15 cytokines are growth hormones such as human growth sulfonates such as Busulfan, Improsulfan and Piposulfan; hormone, N-methionyl human growth hormone, and bovine aziridines such as Benzodopa, Carboquone, Meturedopa, growth hormone; parathyroid hormone; thyroxine; insulin; and Uredopa; ethylenimines and methylamelamines includ- proinsulin; relaxin; prorelaxin; glycoprotein hormones such ing altretamine, triethylenemelamine, trietylenephosphor- as follicle stimulating hormone (FSH), thyroid stimulating amide, triethylenethiophosphaoramide and trimethylolom- 20 hormone (TSH), and luteinizing hormone (LH); hepatic elamine; nitrogen mustards such as Chlorambucil, growth factor; fibroblast growth factor; prolactin; placental Chlomaphazine, Cholophosphamide, Estramustine, Ifosf- lactogen; tumor necrosis factor-a and -(3; mullerian-inhib- amide, mechlorethamine, mechlorethamine oxide hydro- iting substance; mouse gonadotropin-associated peptide; chloride, Melphalan, Novembiehin, Phenesterine, Predni- inhibin; activin; vascular endothelial growth factor; integrin; mustine, Trofosfamide, uracil mustard; nitroureas such as 25 thrombopoietin(TPO); nerve growth factors such as NGF-(3; Cannustine, Chlorozotocin, Fotemustine, Lomustine, platelet growth factor; transforming growth factors (TGFs) Nimustine, and Ranimustine; antibiotics such as Aclacino- such as TGF-a and TGF-(3; insulin-like growth factor-I and mysins, Actinomycin, Authramycin, Azaserine, Bleomycins, -II; erythropoietin (EPO); osteoinductive factors; interferons Cactinomycin, Calicheamicin, Carabicin, Carminomycin, such as interferon-a, -(3 and -y; colony stimulating factors Carzinophilin, Chromoinycins, Dactinomycin, Daunorubi- 30 (CSFs) such as macrophage-CSF (M-CSF); granulocyte- cin, Detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, macrophage-CSF (GM-CSF); and granulocyte-CSF Epirubicin, Esorubicin, Idambicin, Marcellomycin, Mito- (GCSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, mycins, mycophenolic acid, Nogalamycin, Olivomycins, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a Peplomycin, Potfiromycin, Puromycin, Quelamycin, Rodo- tumor necrosis factor such as TNF-a or TNF-(3; and other rubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, 35 polypeptide factors including LIE and kit ligand (KL). As Zinostatin, and Zorubicin; anti-metabolites such as Metho- used herein, the tern cytokine includes proteins from natural trexate and 5-fluorouracil (5-FU); folic acid analogues such sources or from recombinant cell culture and biologically as Denopterin, Methotrexate, Pteropterin, and Trimetrexate; active equivalents of the native sequence cytokines. purine analogs such as Fludarabine, 6-mercaptopurine, Imaging Agent Thiamiprine, and Thioguanine; pyrimidine analogs such as 40 The imaging agent may be any substance that provides Ancitabine, Azacitidine, 6-azauridine, Cannofur, Cytara- imaging information about a targeted site in a body of an bine, Dideoxyuridine, Doxifluridine, Enocitabine, Floxuri- animal, such as a mammal or a human being. The imaging dine, and 5-FU; androgens such as Calusterone, Dromo- agent may comprise magnetic material, such as iron oxide, stanolone Propionate, Epitiostanol, Rnepitiostane, and for magnetic resonance imaging. For optical imaging, the Testolactone; anti-adrenals such as aminoglutethimide, 45 active agent may be, for example, semiconductor nanocrys- Mitotane, and Trilostane; folic acid replenisher such as tal or quantum dot. For optical coherence tomography frolinic acid; aceglatone; aldophosphamide glycoside; ami- imaging, the imaging agent may be metal, e.g., gold or nolevulinic acid; Amsacrine; Bestrabucil; Bisantrene; Eda- silver, nanocage particles. The imaging agent may be also an traxate; Defofamine; Demecolcine; Diaziquone; Elfornith- ultrasound contrast agent, such as a micro or nanobubble or ine; elliptinium acetate; Etoglucid; gallium nitrate; 50 iron oxide micro or nanoparticle. hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitox- Administration antrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; The multistage delivery vehicle may be administered as a Pirarubicin; podophyllinic acid; 2-ethylhydrazide; Procar- part of a composition, that includes a plurality of the bazine; PSKO; Razoxane; Sizofrran; Spirogermanium; vehicles, to a subject, such as human, via any suitable tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; 55 administration method in order to treat, prevent and/or Urethan; Vindesine; Dacarbazine; Mannomustine; Mito- monitor a physiological condition, such as a disease. The bronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside particular method employed for a specific application is ("Ara-C"); cyclophosphamide; thiotEPa; taxoids, e.g., Pacli- determined by the attending physician. Typically, the com- taxel (TAXOL(k, Bristol-Myers Squibb Oncology, Princ- position may be administered by one ofthe following routes: eton, N.7.) and Doxetaxel (TAXOTEREO, Rhone-Poulenc 60 topical, parenteral, inhalation/pulmonary, oral, vaginal and Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-thio- anal. guanine; Mercaptopurine; Methotrexate; platinum analogs Embodiments of the multistage delivery vehicles may be such as Cisplatin and Carboplatin; Vinblastine; platinum; particularly useful for oncological applications, i.e. for treat- etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxan- ment and/or monitoring cancer or a condition, such as tumor trone; Vincristine; Vinorelbine; Navelbine; Novantrone; 65 associated with cancer. For example, skin cancer may be Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandro- treated and/or monitored by topical application of, prefer- nate; CPT-11; topoisomerase inhibitor RFS 2000; difluo- ably, a viscous suspension; lung cancer may be treated US 10,143,658 B2 23 24 and/or monitored by inhalation of an aerosolized aqueous Rohm Pharma. Generally, the enteric coating may be applied microdevice suspension; cervical cancer may be treated from about 0.5% by weight to about 10% by weight of the and/or monitored by vaginal administration of a microdevice tablet or capsule. suspension; and colon cancer may be treated and/or moni- In some embodiments, a less water-soluble polymer, such tored by rectal administration of such a suspension. The 5 as methylcellulose, could be used to delay release of the majority of therapeutic applications may involve some type drug from the chambers following release of the particles in of parenteral administration, which includes intravenous the duodenum. ChronSetRTM technology developed by (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) injection. ALZA Corporation(Mt. View, Calif.) may be used to release Administration ofthe multistage delivery vehicles may be a bolus of the multistage delivery vehicles at designated systemic or local. The non-parenteral examples of admin- l0 times and at targeted sites after passage from the stomach istration recited above, as well as i.m. and s.c. injections, are into the small intestine. In this case, a suspension of the vehicles may be loaded into ChronSet capsules. After swal- examples of local administration. Intravascular administra- lowing, the capsules pass intact through the stomach. The tion may be either local or systemic. Local intravascular shell may be engineered to regulate the rate of water delivery may be used to bring a therapeutic substance to the 15 imbibition the osmotically permeable portion of the system. vicinity of a known lesion by use of guided catheter system, The osmotic engine may expand to push and separate two such as a CAT-scan guided catheter. General injection, such halves of the capsule. The length of the capsule halves may as a bolus i.v. injection or continuous/trickle-feed i.v. infu- be specifically designed to produce separation at pre-se- sion are typically systemic. lected times. The contents of each capsule may be expelled For intravenous administration, the multistage delivery 20 into the intestinal lumen at 2 to 20 hours after administra- vehicle may be formulated as a suspension that contains a tion. Greater than 80% of contents (in this case a suspension plurality ofthe vehicles. Preferably, the vehicles are uniform of drug-filled microfabricated particles) may be expelled in their dimensions and their content. To form the suspen- within 15 minutes time frame. This approach may provide a sion, the vehicles as described above may be suspended in means of releasing the suspension of the vehicles at prese- any suitable aqueous carrier vehicle. A suitable pharmaceu- 25 lected areas of the small or large intestine. Such a system tical carrier is one that is non-toxic to the recipient at the may be used to release the multistage delivery vehicles at dosages and concentrations employed and is compatible sites in the small or large intestine that are optimal for with other ingredients in the formulation. Preparation of binding, such as areas, which contain receptors for the suspension of microfabricated particles is disclosed, for muco-adhesive ligand grafted to the particles, and/or absorp- example, in US patent application publication No. 30 tion, such as regions of the intestinal epithelium that are 20030114366. sensitive to the permeation enhancers contained inside the For oral administration, the multistage delivery vehicle first stage particle. Embodiments described herein are further illustrated by, may administered as a part of an oral composition made up though in no way limited to, the following working of a plurality of the vehicles. Preferably, the vehicles in the 35 examples. composition are uniform in dimensions, i.e. the first stage particles of each vehicle have the same or substantially the WORKING EXAMPLE 1 same dimension; the second stage particles of each vehicle have the same or substantially the same dimensions. The The following experiments were conducted to study load- composition may be made by mixing the vehicles with 40 ing and release of selected second stage particles into suitable non-aqueous carriers, such as oil or micronized nanoporous silicon first stage microparticles. Biodegrada- powder, and filled in unit dose amounts into standard tion and biocompatibility of the nanoporous silicon first enteric-coated capsules or, alternatively, compressed into stage particles was also studied. tablets and coated with an enteric coating material. The Materials and Methods enteric coating ensures that the vehicles are transported in 45 Z2 Analysis dry form through the low (acidic) pH environment of the Particles were counted in a Z2 Coulter® Particle Counter stomach and released at pre-selected regions of the small or and Size Analyzer (Beckman Coulter). The aperture size large intestine. used for particle analysis was 50 µm. The lower and upper The composition may be coated with a protective poly- size limits for analysis were set at 1.8 and 3.6 µm. For mer. This material may be applied by film coating technol- 50 analysis,particles were suspended in the balanced electrolyte ogy to either tablets or capsules to protect the product from solution of the instrument (ISOTON® II Diluent) and the effects of, or prevent release of drugs in, the gastric counted. The total volume of original suspension of particles environment. Such coatings are those, which remain intact did not exceed 0.3% of the final analysis volume. in the stomach, but may dissolve and release the contents of Oxidation of Silicon Microparticles the dosage form once it reaches the small intestine. The 55 Silicon microparticles in IPA were dried in a glass beaker purpose of an enteric coating may be to delay release of the kept on a hot plate (80-90° C.). Silicon particles were first stage particle's content. oxidized in piranha (1 volume H2O2 and 2 volumes of One of the most extensively used enteric polymer may be H2SO4). The particles sonicated after H2O2 addition and cellulose acetate phthalate (CAP). Another useful polymer then acid was added. The suspension was heated to 100-110° may be polyvinyl acetate phthalate (PVAP), which is less 60 C. for 2 hours with intermittent sonication to disperse the permeable to moisture, more stable to hydrolysis and able to particles. The suspension was then washed in DI water till ionize at a lower pH than is CAP. These properties may the pH of the suspension is —5.5-6. Particles were then allow more reliable release in the duodenum. Another transferred to appropriate buffer, IPA or stored in water and example of currently used polymers may be those based on refrigerated till further use. methacrylic acid-methacrylic acid ester copolymers with 65 Surface Modification of Si Particles with APTES acid ionizable groups. Represented among these are poly- Prior to the silanization process, the oxidized particles mers having the tradename Eudragit available through were hydroxylated in 1.5 M HNO3 acid for approximately US 10,143,658 B2 26 1.5 hours (room temperature). Particles were washed 3-5 for fluorescence intensity. FIG. 3, Panel A, presents results times in DI water(washing includes suspending in water and of time dynamics for loading carboxy modified quantum centrifuging, followed by the removal of supernatant and the dots and amino modified quantum dots into APTES modi- repeating of the procedure). fied LP and SP silicon particles. FIG. 4, Panels A-D present The particles were suspended in IPA (isopropyl alcohol) 5 results of time dynamics for loading carboxy modified by washing them in IPA twice. They were then suspended in quantum dots and amino modified dots into (Panel A) LP IPA containing 0.5% (v/v) of APTES (3-aminopropyltri- oxidized silicon particles; (Panel B) LP APTES modified ethoxysilane) for 45 minutes at room temperature. The silicon particles; (Panel C)SP oxidized silicon particles and particles were then washed with IPA 4-6 times by centrifu- (Panel D) SP APTES modified silicon particles. Each of gation and stored in IPA refrigerated. Alternatively, they to Panels A-D also presents time dynamics for loading PEG- were aliquoted, dried and stored under vacuum and dessi- FITC single wall carbon nanotubes, as described below. cant till further use. Determining Concentration of Amino-PEG and Carboxyl Attaching PEG to APTES Modified Particles PEG was attached to the microparticles to provide a Q-dots for Porous Silicon Microparticles Loading spacer for further coupling with anti-VEGFR2 antibody. 15 In a low binding micro centrifuge tube, 3.0x105 LP or SP Fmoc-PEG-NHS, which provided the NHS ester for rapid oxidized silicon or APTES modified microparticles, were coupling to amine groups and an Fmoc group to protect the combined with 0.01, 0.1, 1, 10, 100, 1000 and 2000 nM amine on the PEG, was useed. The 106-109 particles/ml of Amino-PEG or Carboxyl Q-dots respectively, in 200 mM APTES modified microparticles were resuspended in PBS TRIS-HCl pH 7.3. Samples were incubated in a rotating (pH-7.2) and Fmoc-PEG-NHS at a concentration of 1-10 20 wheel (20 rpm) for 15 minutes at 25° C. in a final volume mM added to the particles. of 20 µl for each experimental point. After incubation, the Coupling was carried out for 30 min to 1 h at room samples were diluted with Tris 20 mM,pH 7.3 to 150 µl and temperature, then unreacted Fmoc-PEG-NHS groups were promptly read at a FACScalibur flow cytometer for fluores- washed by centrifugation 3-5 times in PBS. The Fmoc group cence intensity. on the distal end of PEG coupled to the silicon micropar- 25 Determining Time for Loading ofAmino-PEG and Carboxyl ticles were deprotected with piperidine(20% v/v for 30 min) Q-dots into Porous Silicon Microparticles to provide a free amine on the PEG for further coupling with In a low binding micro centrifuge tube, 1.2x106 LP and antibodies. SP oxidized silicon or APTES modified microparticles, were Fluorescent Tagging of Anti-VEGFR2 combined with 2µM Amino-PEG Q-dots or Carboxyl The antibody to VEGFR2 was conjugated with Alexa 488 30 Q-dots in 200 mM TRIS-HCl pH 7.3 in a 80 µl final volume. using an antibody labeling kit from Invitrogen Corp. The Particles and Q-dots were incubated in a rotating wheel (20 fluorescent tag was conjugated to the antibody by following rpm) and sampled out from the vial after 15, 30, 45 and 60 the procedure provided in the manual of the kit. 1 mg/ml of minutes at 25° C. After incubation, the samples were diluted antibody was used for tagging. The amount of antibody with Tris 20 mM, pH 7.3 to 150 µl and promptly read at a conjugated to the fluorescent tag was determined by ana- 35 FACScalibur flow cytometer for fluorescence intensity. lyzing the antibody with a DUO 730 UV/Vis Spectropho- Loading of Amino-PEG and of Carboxyl Q-dots into Oxi- tometer (Beckman Coulter Inc., CA, USA) at 280 and 494 dized and APTES Modified Porous Silicon Microparticles mu. The amount of antibody conjugated was found to be 407 2.1x106 LP silicon microparticles either oxidized or µg/ml. APTES modified were combined with 2µM Amino-PEG Coupling Anti-VEGFR2 to PEG Modified Particles 40 Q-dots or Carboxyl Q-dots respectively, in a TRIS-HCl 200 Silicon particles, resuspended in PBS, were treated with mM pH7.3 solution. Final incubation volume was 140 µl. the heterobifunctional crosslinker ANB-NOS (dissolved in Samples were incubated in a rotating wheel (20 rpm)for 15 DMF and added to a final concentration of 10 mM)for 30 minutes at 25° C. The microparticles were then washed in min to 1 h. The reaction was carried out in the dark to 1.4 mL deionized water (10 folds dilution), and centrifuged prevent the photolysis of the nitrophenyl azide group on the 45 5 minutes at 4200 RPM in a Beckman Coulter Allegra X-22 crosslinker The particles were then washed with PBS to centrifuge. Particles pellet was resuspended in 70 µl of remove unreacted crosslinker and mixed with the fluores- deionized water and 10 µl were taken out the vial, diluted cently labeled anti-VEGFR2. 4.6x106 particles were used with a TRIS 20 mM,pH 7.3 solution to 150 µl final volume. for each experimental point. The different amounts of anti- Sample's fluorescence was immediately read with Becton body used, were 0.814, 0.407, and 0.163 µg, corresponding 5o Dickinson FACScalibur flow cytometer and recorded as to 2.7, 1.35, and 0.54 µg/ml respectively, and exposed to UV time 0 or loading fluorescence. light for 10-15 min to couple the amine groups present on Release of Amino-PEG and of Carboxyl Q-dots from Oxi- the antibody to the attached crosslinker dized and APTES Modified Porous Silicon Microparticles Determining Buffer Cconcentration for Loading of Amino- The residual 60 µl were diluted 10 times into 600 µL of PEG Quantum-dots(Q -dots) into Porous Silicon Micropar- 55 TRIS-HCL 20 mM NaCl 0.9% release buffer. 100 µl were ticles taken out this solution and additionally diluted 5 times by In a low binding micro centrifuge tube, 3.0x105 large pore aliquoting the sample into tubes pre filled with 400 uL (LP) and small pore (SP) oxidized silicon (stored at the TRIS-HCL 20mM NaCI 0.9% release buffer. Final dilution concentration of 300x106 particles/ml) or APTES modified at this point was 500 folds. Samples were put in a rotating microparticles (stored at the concentration of 200x106 par- 60 wheel (20 rpm) for the given amount of time (15, 45, 90, ticles/ml), and 2µM Amino-PEG Q-dots or Carboxyl Q-dots 180, 360 and 1200 minutes) minutes at 37° C. At each time were combined in a solution containing 10, 20, 50, 100 or point the aliquots were centrifuged 5 minutes at 4200 RPM 200 mM of TRIS-HCl at pH 7.3. Samples were incubated in in a Beckman Coulter Allegra X-22 centrifuge. Each pellet a rotating wheel (20 rpm)for 15 minutes at 25° C. in a final was then resuspended in 150 µl of TRIS 20 mM and volume of 20 µl for each experimental point. After incuba- 65 fluorescence was immediately read with a Becton Dickinson tion, the samples were diluted with Tris 20 mM, pH 7.3 to FACScalibur flow cytometer. FIG. 5A presents time dynam- 150 µl and promptly read at a FACScalibur flow cytometer ics of release of amino-modified quantum dots from LP US 10,143,658 B2 27 28 oxidized silicon particles. FIG. 5B presents time dynamics Release of PEG-FITC-SWNTs from Oxidized and APTES of release of carboxy modified quantum dots from APTES Modified Porous Silicon Microparticles modified LP silicon particles. The residual 50 µl were diluted 10 times into 500 µL of Determining Buffer Concentration for Loading of Poly TRIS-HCL 20mM NaCl 0.9% release buffer. 100 µl were (Ethylene Glycol)(PEG) Fluorescein Isothiocyanate(FITC) 5 taken out this solution and additionally diluted 5 times by Conjugated Single Walled Carbon Nanotubes(SWNTs) into aliquoting the sample into tubes pre filled with 400 uL Porous Silicon Microparticles TRIS-HCL 20 mM NaCl 0.9% release buffer. Final dilution In a low binding micro centrifuge tube, 3.0x105 large pore at this point was 500 folds. Samples were put in a rotating oxidized silicon or APTES modified microparticles, were wheel (20 rpm) for the given amount of time (15, 45, 120, io 240 and 1200 minutes) minutes at 37° C. At each time point combined with 20 ng/µl PEG-FITC-SWNTs in a solution the aliquots were centrifuged 5 minutes at 4200 RPM in a containing different molarities (20, 100, 200 mM)of TRIS- Beckman Coulter Allegra X-22 centrifuge. The supernatants HCl pH 7.3. Final volume was 20 µl for each experimental were removed and placed in a 96 well plate for fluorescence point. Samples were incubated in a rotating wheel (20 rpm) reading using the SPECTRAmax plate reader. Each pellet for 15 minutes at 25° C. After incubation, the samples were 15 was then resuspended in 150 µl of TRIS 20 mM and resuspended in 150 µl of TRIS 20 mM and immediately read fluorescence was promptly read with a Becton Dickinson at a FACScalibur flow cytometer for fluorescence intensity. FACScalibur flow cytometer. FIG. 5A presents time dynam- Determining Time for Loading of PEG-FITC-SWNTs into ics of release of PEG-FITC-SWNTs from LP oxidized Porous Silicon Microparticles silicon particles. FIG. 5B presents time dynamics of release In a low binding micro centrifuge tube, 1.2x106 large pore 20 of PEG-FITC-SWNTs from APTES modified LP silicon oxidized silicon or APTES modified microparticles, were particles. combined with 20 ng/µl PEG-FITC-SWNTs in 20 mM Loading of Nanoliposomes into Oxidized andAPTES Modi- TRIS-HCl pH 7.3 in a 80 µl final volume. Particles and fied Porous Silicon Microparticles SWNTs were incubated in a rotating wheel (20 rpm) and 1.5x106 LP and SP oxidized silicon microparticles were sampled out from the vial after 15, 30, 45 and 60 minutes at 25 combined with 10 ng/µl of liposomes, containing an Alexa 25° C. After incubation, the samples were diluted with Tris fluo 555 conjugated siRNA, in a 20 mM TRIS-HCl pH7.3 20mM,pH 7.3 to 150 µl and promptly read at a FACScalibur solution. Particles and nanoliposomes were incubated in a flow cytometer for fluorescence intensity. rotating wheel (20 rpm) and sampled out from the vial after Determining Concentration of PEG-FITC-SWNTs for 15, 30 and 60 minutes at 25° C. After incubation, the Porous Silicon Microparticles Loading 30 samples were diluted with Tris 20 mM,pH 7.3 to 150 µl and In a low binding micro centrifuge tube, 3.0x105 LP or SP promptly read at a FACScalibur flow cytometer for fluores- oxidized silicon or APTES modified microparticles, were cence intensity. combined with 1, 10, 20 and 50 ng/µl PEG-FITC-SWNTs in Degradation 20 mM TRIS-HCl pH 7.3 in a final volume of 20 µl for each 5x106 LP and SP oxidized silicon particles were either experimental point. After incubation, the samples were 35 mixed in a solution containing 2.5 mM TRIS and 0.9% NaCl diluted with Tris 20 mM,pH 7.3 to 150 µl and promptly read at pH 7.3 (Saline) or in cell culture media supplemented with at a FACScalibur flow cytometer for fluorescence intensity. 10% FBS (CCM). The mixture was incubated in a rotating Determining Fluorescence Intensity and Quenching Effect wheel (8 rpm) at 37° C. and sampling for SEM,Z2 and ICP of PEG-FITC-SWNTs Using Fluorimetry was performed at time 0 and after 6, 18 and 24 hours. A concentration curve for PEG-FITC SWNTs in 20 mM 40 The experiment was designed in order to have enough TRIS-HCL pH 7.3 was determined using a SPECTRAmax material to perform the three types of analysis for the fluorimeter. A serial dilution was performed, starting with 35 degradation study in parallel at the same time, thus avoiding ng/µl and proceeding by a factor of 1:2 down to minimum differences due to uncontrolled or unnoticed variables. This detectable amount of 107 pg/µl. The aliquots were placed in protocol therefore applied to the samples obtained for SEM, a 96 well plate and the fluorescence was read using excita- 45 Z2 and ICP. tion at 485, and emission at 520. Fluorescence quenching An additional degradation experiment was performed to was observed at the higher concentrations of SWNTs, as provide material for the toxicity experiment with HUVEC shown by their lower fluorescence. cells. For each experimental point. we incubated 3x106 LP, Loading of PEG-FITC-SWNTs into Oxidized and APTES oxidized or Aptes modified, or SP, oxidized or Aptes modi- Modified Porous Silicon Microparticles 50 fied, silicon particles in CCM in a rotating wheel (8 rpm) at 1.8x106 "large pore" (LP, approximately 30 mu) silicon 37° C. for 24. microparticles were combined with 20 ng/µl PEG-FITC- ICP SWNTs in a 20 mM TRIS-HC1 pH7.3 solution. Final incu- To understand whether the porous Silicon particles dis- bation volume was 120 µl. Samples were incubated in a appeared from the solutions, they were incubated in, the rotating wheel (20 rpm) for 15 minutes at 25° C. The 55 porous Silicon particles were dissolved in Silicic Acid and microparticles were then washed in 1.2 mL deionized water studied by a technique called Inductive Coupled Plasma (10 folds dilution), and centrifuged 5 minutes at 4200 RPM Atomic Emission Spectrometry (ICP-AES). in a Beckman Coulter Allegra X-22 centrifuge. After incu- This methodology allows quantifying the absolute bation, the supernatants were removed and placed in a 96 amount of any given element present in a solution. Amples well plate for fluorescence reading using the SPECTRAmax 60 were withdrawn at the same time points, the Z2 and the SEM plate reader. analysis and centrifuged them to pellet down the particles Particles pellet was re-suspended in 60 µl of deionized that were not degraded yet. The surnatants filtered through water and 10 µl were taken out the vial, diluted with a TRIS a 0.450 µm filter unit were analyzed with ICP. 20 mM, pH 7.3 solution to 150 µl final volume. Sample's Cell Culture fluorescence was immediately read with Becton Dickinson 65 Half a million freshly isolated Human Umbelical Vein FACScalibur flow cytometer and recorded as time 0 or Endothelial Cells(HUVEC) (CloneticsTM Cambrex Bio Sci- loading fluorescence. ence Walkersville, Inc) were plated in M199 medium US 10,143,658 B2 29 30 (Gibco/Life Technologies Inc.) supplemented with 10% while as a negative control we added 50 µg/ml of Cis- foetal calf serum (FCS, Gibco), containing 100 IU/ml peni- platinum to cell culture media. cillin (Sigma), 100 g/ml streptomycin (Sigma), 7.5 IU/ml The plates were incubated at 37° C. and, at the time points heparin (Sigma), 2 ng/ml epidermal growth factor (EGF) of 12, 24, 48 and 72 hours, the medium was removed from (R&D system) and 250 pg/ml endothelial cell growth factor 5 the well and the cells extensively washed with sterile PBS to (-ECGF) (BioSource, USA), referred to as complete remove Silicon particles. The proliferation assay was then medium. Confluent cells were detached with trypsin (Sigma) performed as described for the calibration assay. All the for subculturing. The cells were expanded until passage 6 experiments were done in triplicate and results expressed as and used for the biocompatibility studies. mean values with standard deviations. Microscopy 10 Propidium Iodide Staining Cells were analyzed in bright field contrast with an 0.6x106 cells were seeded in 25 cm2 flasks and when the Olympus CKX41 microscope with a 40x magnification lens. cells were attached (approximately 6 hours later, when the Images were taken with an SP-350 Olympus True-Pic cells generated a 60% confluent cell bed), 3x106 of either LP TURBO Image Processor camera. 15 oxidized, SP oxidized, LP APTES or SP APTES Silicon LDH Toxicity Assay particles (1:5 ratio)resuspended in 20 µl of sterile, deionized For the calibration of the toxicity assay, different amount H2O, were added to the medium of each flask. As a positive of cells (125, 250, 500, 1000, 2000) were seeded in three 96 control 20 µl of sterile, deionized H2O were added to the well plates. After 12, 24 and 48 hours the cell culture media cells while as a negative control we added 50 µg/ml of was replaced with fresh media containing 1% Triton. The 20 Cis-platinum to cell culture media. As an additional control, cells were incubated at room temperature (RT) for 10 the same amount of HUVEC cells was seeded in 75 cm2 minutes then spun down for 3 minutes at 1200 rpm and 100 flasks to allow them to freely grow without reaching con- µl of cell culture media were transferred into a replica plate fluency during the experiment. kept on ice. 100 µl of a solution containing the reconstituted After 12, 24, 48 and 72 hours cells were washed 3 times catalyst and the dye (Biovision LDH Cytotoxicity Assay 25 with 5 ml of sterile PBS then were trypsinized, harvested Kit) was added to each well and the plate was transferred on and centrifuged. After an additional washing step with PBS, an orbital shaker, covered with foil and shaked at RT for 20 each cell pellet was resuspended in 250 µl of PBS and 750 minutes. The plate was then transferred in a SPECTRAmax µl ofice-cold methanol were added to cell suspensions while fluorimeter and absorbance at 490 mn was read. All the gently vortexing. Fixation was carried on for 20 minutes experiments were done in triplicate and results expressed as 30 then the cells were spun down and washed twice with PBS. mean values with standard deviations. A solution containing (50 µg/ml propidium iodide in 10 mM For the experiment, 1000 cells were seeded and, after the Tris, 5 MM MgC12, pH 7.3 and 75 µg/ml RNAse) was then cells attached on the surface (approximately 6 hours), dif- added to the cell pellet and the suspension was incubated in ferent amount of LP oxidized, SP oxidized, LP APTES and dark on a rotating wheel (5 rpm) for 60 minutes. Samples SP APTES Silicon particles (1000, 5000 and 10000, corre- 35 were then transferred into FACS tubes and immediately read sponding to a 1:1, 1:5, 1:10 cells:particles ratio)resuspended with a Becton Dickinson flow cytometer. in 2µl of sterile, deionized H2O were added to the media. As Flow Cytometry Setup a positive control, 2µl of sterile, deionized H2O were added Particles were assessed for fluorescence using a FACS to the cells while as a negative control we added 50 µg/ml Calibur (Becton Dickinson). Bivariate dot-plots defining log of Cis-platinum to cell culture media. 40 SSC versus log FSC were used to evaluate the size and shape The plates were incubated at 37° C. and, at the time points of the Silicon particles (3 microns in diameter, 1.5 in height) of 12, 24, 48 and 72 hours, the toxicity assay was performed used and to exclude from the analysis non-specific events. as described for the calibration assay with the exception of Control rainbow BD CalibriteTM beads (3.5 micron in size) Triton addition. All the experiments were done in triplicate were analyzed as a standard. and results expressed as mean values with standard devia- 45 Apolygonal region (R1) was defined around the centre of tions. the population excluding the events that were to close to the MTT Assay noise signal of the instrument. For each sample, the number For the calibration of the MTT assay, different amount of of particles detected in the RI region was above 80%. cells (125, 250, 500, 1000, 2000) were seeded in three 96 For mean fluorescence intensity (MFI) determination, log well plates. After 12, 24 and 48 hours the cell culture media 5o FLl and FL2 versus log FSC dot-plots were created by was replaced with fresh media containing 50 µl of MTT dye. gating on the events falling within the defined region (RI). The cells were incubated at 37° C.for 4 hours then the media The peaks generated by each of the samples were analyzed and MTT were removed and 200 µl of DMSO and 25 µl of in the corresponding fluorescent histogram and the geomet- a 0.1M glycine, 0.1 M NaCl, pH 10.5, solution were added ric mean values recorded. to each well and the plate was incubated at RT for 10 55 For particles detection, detectors used were forward scat- minutes. The plate was then transferred in a SPECTRAmax ter(FSC) E-1 and side scatter (SSC) with 474V voltage set. fluorimeter and absorbance at 570 mn was read. All the The fluorescent detector FLl was set at 800V. experiments were done in triplicate and results expressed as Green fluorescence (FITC and Q-dots525) was detected mean values with standard deviations. using FLl, 530/30 mn band-pass filter. Orange and red For the experiment 1000 cells were seeded and, after the 60 fluorescence (Q-dots D565 and Propidium Iodide) was cells attached on the surface and generated a 50% confluent detected using FL2. As single color detection only was being cell bed (approximately 6 hours), different amount of LP analyzed compensation was set at zero. Instrument calibra- oxidized, SP oxidized, LP APTES and SP APTES Silicon tion was carried out before, in between and after each series particles (1000, 5000 and 10000, corresponding to a 1:1, 1:5, of acquisition using rainbow BD Calibrite m beads. 1:10 cells:particles ratio) resuspended in 2 gl of sterile, 65 For cells detection, detectors used were linear forward deionized H2O were added to the media. As a positive scatter (FSC) E00 with an amplification gain of 4.59 and control, 2µl of sterile, deionized H2O were added to the cells linear side scatter (SSC) with the voltage set at 474V with US 10,143,658 B2 31 32 an amplification gain of 1.07. The fluorescent detectors FL2 TABLE 5-continued was set at 449V with the amplification gain set at 1.32. Cell and particle debris were electronically gated from the Zeta Potential of microparticles and analysis on basis of scatter properties. In all the experiments nanoparticles used in loading experiments at least 20,000 particles or cells were analyzed. All the 5 Size of Zeta potential Buffer conidition experiments were performed in triplicate. The results are Particle nanopores (nn) (mV) at pH-7.3 presented as mean fluorescence intensity of intact particles or viable cells only. Data analysis was carried out with LP MPTMS modified Si 20-40 —16.9 20 mM Tris —6.17 200 mM Tris CellQuest software (BD Biosciences). SP oxidized Si 5-7 —11.15 20 mM Tris Fluorescent Microscopy 10 —13 200 mM Tris Fluorescent imaging of particles was performed with a SP APTES modified Si 5-7 6.42 20 mM Tris Nikon Eclipse TE2000-E with a DQC-FS Nikon CCD —7.42 200 mM Tris Camera kept at —30.1° C. All the samples were mounted SP MPTMS modified Si 5-7 —18.3 20 mM Tris —8.21 200 mM Tris immediately before the analysis and the images acquired Amino gDots NA —1.21 20 mM Tris with a 63x immersion oil objective. The microscope settings 1.3 200 mM Tris 15 were kept constant throughout all the experiments. Numeri- Carboxy gDots NA —32.8 20 mM Tris cal Aperture was set at 1.4, Refractive Index at 1.515, —10.1 200 mM Tris Exposure Time at 500 ms, Readout Speed at 10 MHz and SWNTs NA —9.21 20 mM Tris Conversion Gain at 1/6x. The images were analyzed and measured with the NIS Elements AR 2.3 Software. Load and Release 20 Results Quantum dots (Qdots) and single-wall carbon nanotubes Antibody Conjugation (SWNT) were used as second stage nanovectors for their The total number of particles used for conjugating APTES distinct and excellent properties. Q-dots are light-emitting modified particles with the anti-VEGFR2 was —7.03x106. semiconductor nanocrystal (CdSe or CdTe), and many Two different amounts of anti-VEGFR2 were used for the research has been conducted on in-vitro and in-vivo molecu- conjugation as listed in Table 3 below. 25 lar and cellular imaging because of their high brightness, photostability and the capacity of multiplexed color coding. TABLE 3 Q-dots were coated with hydrophilic polyethylene glycol Amount of anti-VEGFR2 added to particles (PEG) to increase biocompability. The zeta potential was -1mV for Amino-PEG Qdots in 20 mM Trs buffer, and —32.8 30 Anti-VEGFR2 Anti-VEGFR2 per 106 mV for carboxyl PEG Q-dots. The hydrodynamic size was Particles ID added (µg) particles (µg) measured by the diffusion coefficient of colloidals in solu- APTES 1 2.04 0.29 tion using scattering light dynamic light scattering tech- APTES2 0.407 0.058 nique. Amino and Carboxyl Q-dots show similar hydrody- namics size (13 and 17 nm respectively). 35 In a different experiment, after conjugation, the particles SWNT is well-ordered hollow "rolled-up" structures, and were washed and centrifuged in phosphate buffer containing has high aspect ratio, high surface area, high mechanical 0.5% Triton X-100 6 times followed by 4 washes in plain strength, ultra light weight, excellent chemical/thermal sta- phosphate buffer and then read on the plate reader. Two bility. Many protocols have been studied to functionalize the different amounts of anti-VEGFR2 were used for the con- SWNT surface with peptides, proteins and drugs. In this jugation as listed in Table 4 below. study, SWNT was of diameter 2-4 nun and lengths between 30 and 100 mu, the surface was functionalized with PEG- TABLE 4 amine, providing solubility in water and physiological saline and further linked to FITC for fluorescent imaging. The zeta Amount of anti-VEGFR2 conjugated to particles as detected by potential measurement shows —9.2 mV in 20 nM TRIS plate reader based on fluorescence assay of the anti-VEGFR2. 45 buffer. The effect of Tris concentration on the loading of quantum Particles No. of Amt. of Anti-VEGFR2 Anti-VEGFR2 per ID particles (µg) 106 particles (µg) dots in large pores particles was first investigated. Tris was selected as a buffering agent because, it may give the highest APTES1 7.42 x 105 0.065 0.088 stability to the quantum dots (Q-dots) compared to other APTES2 7.42 x 105 0.022 0.03 50 buffering agents. The mean fluorescence of Q-dots loaded particles was studied as a function of Tris concentration. The As indicated by Table 4, there was a significant reduction fluorescence signal on the particles increased as the Tris in the number of particles in all the cases during the concentration increases. The concentration of 200 mM Tris conjugation. This might be due to losses incurred during the gave the best effect in terms of loading and was therefore selected for all the subsequent experiments. numerous washing steps involved in removing unbound 55 antibody from the solution. The loading capacity of "large porous" silicon particles was evaluated by using "small porous" silicon particles as TABLE 5 the control. The hydrodynamic size of Q-dots is 13-15 mu, and should not be able to get into the small pores (<5 mu) Zeta Potential of microparticles and but stay on their surface. The concentration effect of Q-dots nanoparticles used in loading experiments for the loading was performed by changing the amount of Size of Zeta potential Buffer conidition Q-dots from 10 picomolar to 2 micromolar and incubating Particle nanopores (nn) (mV) at pH-7.3 all the reactions in the same final volume(20 µl ). Sample's loading was analyzed at FACS and the quantum dot con- LP oxidized Si 20-40 —10.1 20 mM Tris 1.31 200 mM Tris centration of 2µM resulted in the highest level of loading LP APTES modified Si 20-40 6.52 20 mM Tris and therefore used for all the subsequent experiments. 5.19 200 mM Tris The loading time/dynamics of particles with Q-dots were studied by different incubation time (15 min, 30 min and 60 US 10,143,658 B2 33 0_1 min). The loading of Q-dots in the particles was a very fast duration of the experiment and reached its maximum at the process that reached its maximum within 15 min. After that, 60 minutes time point (FIG. 4, Panel A, and FIG. 21). the fluorescence signal drops slowly with time as shown in The same loading patterns were occurring during the FIG. 21, Panels a-d. loading of small pore particles (SP). The APTES modified The surface properties of particles also proved to be 5 particles loaded very quickly and then lost some of their important in the loading kinetics. As shown in FIG. 21, associated fluorescence through time (FIG. 4, Panel D, and Panels a-d, carboxyl PEG Q-dots showed little loading into FIG. 21), while the oxidized SP loading increased over time the oxidized particles. This may be attributed to the elec- as happened with the LP particles suggesting that APTES trostatic force since both particles were negatively charged. modification played a crucial role in the loading process io (FIG. 4, Panel C, and FIG. 21). When the surface of the silicon particles was modified with The kinetics of PEG-FITC-SWNTs release from LP oxi- amino groups (APTES), the zeta potential became positive dized particles were exponential like in the first time points. (+5—+6 mV) as shown in Table 5. The loading of carboxyl The release slowed down in later time points. After 20 hours Q-dots significantly increased into APTES treated particles. only a residual 20% of the initial fluorescence was found The charge of amino-PEG Q-dots is almost neutral (zeta 15 associated to the particles suggesting that some of the potential —1—+1 mV), and the APTES treatment of silicon payload could be retained into the nanoporous silicon par- particles had only a minor impact on the loading (zeta ticles (FIG. 7B and FIG. 21) potential changed from —11 mV to +6 mV). Nanoliposomes also loaded into LP particles with the The kinetics of amino quantum dots release from LP same dynamics as described above. The fluorescence asso- oxidized particles were exponential like with a 33% 20 ciated with the Silicon particles were visualized by means of decrease of the amount of loaded nanoparticles after 20 fluorescence microscopy (FIG.8A, Panels A and B, FIG. 813, minutes and of 67.5% after 45 minutes. After 20 hours only Panels E and F). a residual 1% of the initial fluorescence was found associ- Degradation ated to the particles demonstrating that all the quantum dots Porous Silicon (PSi) is known to be fully biodegradable could be released from the nanoporous silicon particles. 25 (Mayne 2000; Low, Williams et al. 2006) and biocompatible Fluorescence Quenching (Canham 1995). The oxidation process, that was performed The fluorescence of a solution containing 35 µg/ml of according with protocols already described for Silicon FITC conjugated single wall carbon nanotubes (FITC- wafers(Canham 1997), introduced hydroxil groups(OH) on SWNTs) and of a serial dilution of them (ranging from 1:2 the exposed Silicon surface and these groups, in the presence to 1:2048) in milliq pure water was measured (FIG. 6A, 30 of water, are subjected to hydrolysis thus converting solid Panel A). The fluorescence values of the first 3 and more Silicon into highly soluble Silicic acid. concentrated samples (respectively 35, 17.5 and 8.725 SEM µg/ml) were lower than the values of less concentrated The study of the modifications in the size, shape and solution. This is a phenomenon known as fluorescence overall aspect of the particles was performed by Scanning quenching. As a control the same readings were performed 35 Electron Microscopy (SEM)(FIGS. 9 and 10). The images with a series of solutions of carboxy pegylated quantum dots showed that the particles degraded along time starting from ranging from 8µM to 4 nM that showed a linear decrease of the areas with high porosity and then degradation spread to the fluorescence value along with the dilution. The fluores- particles edges where the porosity is lower and the size of cence quenching dynamics in the loading of APTES modi- the pores smaller. It is suprising to notice that the overall fied LP particles with FITC-SWNTs was evaluated. The 40 shape of the particles remained almost intact until the end of experiments were performed as previously described. The the degradation process occurred. Although the present profiles ofthe curves suggested that the loading ofthe higher inventions arenot limited by a theory of their operation, this amount of FITC-SWNTs induced fluorescence quenching surprising observation may be explained by the fact that thus resulting in decreased fluorescence of the silicon par- corrosion of oxidized Silicon surfaces progressively con- ticles. The concentration of 20 µg/ml was selected for all the 45 sumed the walls in between the pores thus creating bugs and subsequent experiments (FIG. 6A, Panel B and FIG. 20, holes in the center of the particles. The same process took Panel B). more time to dissolve the sides of the particles, where the The effect of Tris concentration on the loading of PEG- number of pores is smaller and the walls between the pores FITC-SWNTs and q-dots in large pores particles was also are thicker. investigated, see FIG. 613, Panels C-F. Panel E and Panel F 50 Z2 Coulter show the mean fluorescence of PEG-FITC-SWNTs loaded The characterization of nanoporous Silicon particles deg- particles in LP APTES modified and LP oxidized silicon radation was performed through several techniques. The particles as a function of Tris concentration. The fluores- measurement of a decrease in the number of particles and in cence signal on the particles decreased as the Tris concen- their volume was performed with Z2 Coulter® Particle tration increased. The concentration of 20 mM Tris gave the 55 Counter and Size Analyzer (Beckman Coulter). The par- best effect in terms of loading and was therefore selected for ticles were kept for 24 hours in a rotating wheel (8 rpm) at all the subsequent experiments. 37° C. in two different solution: TRIS 2.5 mM,0.9% NaCl Carbon Nanotubes Loading at pH7.3 (Saline) and Cell Culture Media with 10% FBS The dynamics of the loading of both oxidized and APTES (CCM). To understand, if the size of the pores had a role in modified LP and SP particles with FITC-SWNTs was evalu- 60 the degradation kinetics, both Silicon particles with smaller ated. The experiments were performed as previously pores of 7-10 (SP) nanometers and particles with larger described using 20 µg/ml of fluorescent nanoparticles (see pores of 20-30 nanometers (LP) were used. FIG. 4, Panels A-D). In this experimental setting, the loading The particles incubated in Saline did not show any time dynamics were fast for the LP APTES particles that significant decrease in their median size distribution, either showed a similar decrease at later time points (FIG. 4, Panel 65 in the case of LP and in the SP. On the contrary, their number B, and FIG. 21). A different kinetic was observed for the LP went progressively down throughout time (FIG. 11, Panel oxidized particles loading that increased during the whole A). These results combined together may mean an overall US 10,143,658 B2 M 36 loss of Silicon particles mass of 50% or more for both LP showing that porosity and surface chemistry did not either and SP particles at 24hrs, as indicated in FIG. 11, Panel C. increased or decreased cell toxicity. All together these results In the experiment performed with particles incubated in did not suggest any massive toxicity due to exposition of CCM,a decrease in both their median size and number(FIG. cells to particles. 11, Panel B). These results combined together indicated a 5 MTT Proliferation Assay loss of particles mass of 60% for the LP particles and more MTT is a yellow water-soluble tetrazolium dye that is than 90% for SP particles. reduced by live cells to a water insoluble purple formazan. ICP The amount offormazan may be determined by solubilizing The readings at the ICP showed a linear increase in the it in DMSO and measuring it spectrophotometrically. Com- amount of silicon present after degradation. These data io parisons between the spectra of treated and untreated cells confirmed the ability ofthe ICP-AES method for verification may give a relative estimation of cytotoxicity. (Alley et al. of degradation. The data were regular, with low standard (1988)Cancer Res. 48:589-601). A toxicity calibration curve deviations, and had a strong correlation with the Z2 readings (FIG. 15, Panel A)to correlate the absorbance values read at of particles count and size during the test. The increased fluorimeter with the number of growing cells. From this degradation that occurred in cell culture media as opposed to 15 calibration curve, the amount of cells that were metaboli- TRIS buffer suggested high biodegradability of these par- cally active was derived. ticles in cell culture conditions (FIG. 12A, Panels A-B and At 12, 24 and 48 hours, the proliferation of cells incubated FIG. 12B, Panels C-D). with particles was exactly the same that in the control plate Biocompatbility (FIG. 15, Panel B). At 72 hours the mean absorbance at 570 Silicon nanoporous particles induced cell toxicity by 20 mu was 1.635 for HUVEC cells cultured without any means of Lactate Dehydrogenase (LDH) toxicity assay particles (positive control) while it was 1.623 from cells (Biovision Inc.), cell proliferation by means of MTT assay incubated with a 1:1 ratio. At higher cells:particles ratios (Promega) and apoptosis and cell cycle by means of Pro- (1:5 and 1:10) HUVEC gave an absorbance value equal to pidium Iodide staining. 1.46 and 1.41 respectively (FIG. 15, Panels B-F). These Bright Field Microscopy 25 lower values correlated with a slight decreased in cell Cells grown in the presence of porous Silicon particles did proliferation when the cells were cultured in presence of a not show any significant or abnormal type of morphological high number of particles. This was in accordance with the change (FIG. 13, Panels A-D)during time. The cells were at little increase in toxicity measured with the LDH assay at 72 50% confluency after 12 hours and they reached almost hours (FIG. 14A, Panel B and FIG. 14B, Panels C-F). 100% confluency after 24 hours. Some signs of apoptosis or, 30 The same proliferation pattern was observed in all the more generally, of cell death were visible at 48 hours and experimental conditions thus showing that porosity and even more at 72 hours (bright, rounded, reflectant cells or surface chemistry did not either increased or decreased cell cells with large, multilobular nuclei or with high narrowing growth. All together these results did not suggest any of the cell body)as indicated by the white arrows in the FIG. significant change in cell viability due to exposition of cells 13,Panels A-D. This was mainly due to the fact that the cells 35 to particles. had already reached confluency and could not find any space Cell Cycle and Apoptosis to grow further and thus underwent some processes of cell HUVEC cells exposed for 12, 24, 48 and 72 hours to a 1:5 degradation. ratio of particles (LP and SP either oxidized or APTES LDH Toxicity Assay modified) were analyzed after fixation and propidium iodide LDH is a cytoplasmic enzyme that is released into the 40 staining at FACS. The forward scattering (FSC) and side cytoplasm upon cell lysis. The LDH assay, therefore, is a scattering(SSC) in a dot plot and in a contour plot were first measure of membrane integrity. The basis of the LDH assay analyzed (FIGS. 16A-1-FIGS. 16C-3, Panels A-C). FSC are oxidization of lactate to pyruvate by LDH, pyruvate parameter provided an information of the size of the cells reaction with the tetrazolium salt to form formazan, and the while SSC provided an information of the shape of the cells. spectrophotometrical detection of the water-soluble forma- 45 The 3D plot on the bottom of each panel indicated on the zan. (Decker, T. & Lohmann Matthes, M. L. (1988) J. z-axis the counts or events and provided an information on Immunol Methods 15:61-69; Korzeniewski, C. & Calle- the overall distribution of the cells analyzed at FACS. waert, D. M.(1983) J. Immunol Methods 64:313-320). The peaks of necrotic/apoptotic cells that were character- A toxicity calibration curve (FIG. 14A, Panel A) was ized by a smaller and less homogeneous shape are indicated made to correlate the absorbance values read at fluorimeter 50 with the red arrows. The data reported in FIGS. 16A-1- with the number of cells that were lysed by 1% Triton FIGS. 16C-3, Panels A-C, are relative to HUVEC cells incubation. From this calibration curve, the amount of cells incubated with Saline (control) and with LP and SP oxidized that were releasing the LDH enzyme during particles incu- Silicon particles. Cells distribution along time changed bation was derived. At 12 and 24 hours, the toxicity signal significantly, with a major accumulation in the low SSC,low was very low and could be mainly due to unbound cells that 55 FSC region at 48 and 72 hours. No significant difference in did not recover from trypsinization and seeding(background the distribution of cells among the 3 groups was noticed and toxicity). This was confirmed by comparing the toxicity the same results were obtained from the cells treated with LP values coming from the experimental plates with those and SP APTES modified particles. Although the present measured in the control plates (FIG. 14A, Panel B). The inventions are not bound by their theory of operation, the toxicity of HUVEC cells grown in the 96 well plates without 60 induction of cell death at later time points could be attributed any particles (positive control) showed at 72 hours a mean to overconfluency. This could be expected since the seeded absorbance of 0.496 at 470nm. That was almost the same cells were at 60% confluency at day 0 in order to keep the value as from cells incubated with a 1:1 ratio (0.507). At the experiment as close as possible to a physiologic solution, in higher cells:particles ratios (1:5 and 1:10) the mean values which the particles would contact the endothelial walls of for toxicity were 0.55 and 0.57 respectively (FIG. 14A, 65 capillaries. Panel B and FIG. 14B, Panels C-F). The same toxicity The distribution of the treated HUVEC cells in the pattern was observed in all the experimental conditions thus different phases of the cell cycle was also measured quan- US 10,143,658 B2 37 38 titatively (FIG. 17A-17D). By comparing the data coming greater stability of the active agents, while providing from control cells (FIGS. 16A-1-FIGS. 16C-3, Panel A, increased delivery and concentrated dosage of therapeutic HUVEC+Saline) with the ones coming from LP and SP agents to a targeted tissue. treated cells it was confirmed that exposure to Nanoporous The multiple and simultaneous loading of second stage Silicon Particles was not toxic and did not induce any 5 nanoparticles into first stage silicon nanoporous particles significant increase in the amount of cell death. It was also was achieved. The second stage particles (Q-dots and demonstrated that there were no alterations in the cell cycle SWNTs) were slowly released over time periods sufficient phases between treated and untreated cells. for the first stage particle to reach a specific biological target In order to demonstrate that not only the intact particles, site. The main physical, chemical and electrostatic mecha- but also their degradation products did not induce any nisms that govern the loading and release processes were. toxicity, HUVEC cells were incubated with CCM containing The nanodelivery system was able to locally release its the degradation product of all the particles type and con- payload into cells. firmed the results described so far (FIG. 17D). A slight Results increase was measured in the amount of cells in the apop- 15 First and Second Stage Particles totic region when the cells were incubated for 72 hours with Silicon microparticles were characterized by Z2 Coulter® the degradation product of SP oxidized and SP and LP Particle Counter and Size Analyzer to measure their volume, APTES modified particles. It may be possible that smaller size and concentration and by Scanning Electron Micros- Silicon fragments produced during the degradation process copy(SEM) to evaluate their morphology in detail. FIG. 19, toxicity45-4a induced some kind of 20 Panels a and b, show representative SEM images of the back and front sides and of the cross sections of"large pore"(LP) WORKING EXAMPLE 2 and "small pore" (SP) silicon particles respectively. These particles are highly porous and hemispherical in shape. The A multi-stage delivery system based on biodegradable diameter of LP particles is approximately 3.5 µm, with pores silicon particles containing nanopores of specific size as first 25 of straight profile, which cross the particles perpendicularly stage carriers that may load, carry, release and deliver into to their external surface and have pore size ranging from 20 cells multiple types of nanoparticles with a precise control to 30 nm (FIG. 19,Panel a). SP silicon particles have a mean was developed. The first stage silicon nanoporous particles diameter of 3.2 µm, and have a flatter shape than LP silicon may be simultaneously loaded with different types of second particles as a consequence of anodization and electropolish- stage nanoparticles, which are released in a sustained fash- 30 ing processes used to produce them, with pore sizes less than ion over time. The major physical, chemical, and electro- 10 nm (FIG. 19, Panel b). Both LP and SP silicon particles static mechanisms that control the loading and release of have a thickness of 0.5-0.6 µm. According to BET analysis, second stage nanoparticles were defined. Finally, it was the surface area is 156 m2 g-1 for LP particles and 294 m2 1 shown that the porous silicon carriers are able to locally 35 g'- for SP particles. Pore volume is 0.542 cm3 g for LP delivery the second stage nanoparticles into the cytoplasm. particles and 0.383 cm3 g-1 for SP particles. The pore Taken together, these studies provide evidence that silicon density, size, shape and profile may be finely tuned by nanoporous particles may be used as cargoes for the simul- changing electrical current, etching time, and doping, which taneous deliver of different types of nanovectors into cells. is reproducible from batch to batch. Non-oxidized silicon is This system may offer unprecedented methods to achieve 40 a hydrophobic material but a variety of surface treatment intracellular delivery of multiple therapeutics and/or imag- protocols are available for silicon-based materials to be ing agents. stabilized and further functionalized with oligonucle- Introduction otides27, biomolecules28, antibodies29'30, and polyethylene Since its development in the last decade, nanotechnology glycol (PEG) chains31. Oxidized silicon particles have a has being used in a wide variety of applications in biomedi- 45 negative zeta potential value (-10.1 for LP; -11-25 for SP) cal research for disease detection, diagnosis, and due to the presence of the hydroxyl groups on the surface of treatment'-3 . The objectives of many studies involving the the particles32. The particles that were modified with APTES development and refinement of nanoparticles have focused have a positive zeta potential value (6.52 for LP; 6.45 for on their use as agents for delivery of therapeutic or imaging SP)as a consequence of the introduction of amino groups on 5 molecules to organs, tissues, and cells4° . A number of 50 their surface. The hydrodynamic size of Q-dots was mea- different nanovectors have been evaluated for therapeutic sured by the diffusion coefficient of colloidal in solution uses, including liposomes of various sizes and composi- using dynamic light scattering technique. Diameter was 13 tion6,7 , quantum dots (Q-dots)$°9, iron oxide10, single wall mu for Amino-PEG Q-dots, and 16 mu for Carboxyl Q-dots. carbon nanotubes (SWNTs)11, gold nanoshells12,13and sev- Size of PEG-FITC-SWNTs was evaluated by AFM micros- eral other types of nanoparticles 14. The progress thus far has 55 copy. The diameter of non PEGylated SWNTs is around 1 given rise to the field defined as "molecularly targeted mu but due to the PEG surrounding the nanotubes, PEG- therapeutics"15-1a however the accomplishment of the FITC-SWNTs used for this study had a mean diameter of 4 original objective, which is to selectively deliver a thera- mu, and a mean length of 30 mu. The SWNTs were peutic agent using a nanovector-based delivery system, has functionalized using PEG-amine through carboxylic acids 19,20 not been fully realized 60 obtained via the SWNT cutting process and were conjugated The integration of nanovectors into nanoporous silicon to FITC to allow for their fluorescent imaging. Second stage particles may allow the nanovectors to evade the natural nanoparticles with fluorescent properties were selected to biological barriers that may normally retard their therapeutic quantify loading into and release from silicon nanoporous effects. Such approach may provide protection of therapeu- particles using established techniques, such as flow cytom- tic agents from surrounding fluids and molecules that may 65 etry, fluorimetry, as well as both fluorescence and confocal normally degrade them prematurely and from uptake by the microscopy. Physical and chemical of first and second stage Reticulo Endothelial System (RES). This may lead to a particles are summarized in Table 6. US 10,143,658 B2 39 40 TABLE 6 Releasing Second Stage Nanoparticles from First Stage Silicon Particles Chemical Pore Zeta To evaluate the multistage delivery system as a tool for I" Stage Particle Modification size, nm potential, mV drug delivery, the kinetics of release of the second stage "Large pore" (LP) oxidized 20-40 —10.1 particles from the nanoporous silicon first stage particles LP APTES 20-40 6.52 was investigated. In order to mimic physiologic conditions, "Small pore" (SP) oxidized 5-7 —11.15 all the experiments were performed at 37° C. in buffered SP APTES 5-7 6.42 saline and the release solution was replaced at each time

2"d Stage Particle Size, nm point. FIG. 21, Panels e-h, demonstrate that both types of 10 second stage particles were released over time from the first Q-dots Amino PEG 13 —1.21 stage silicon particles. Surprisingly, the release process was Q-dots Carboxyl PEG 16 —32.8 SWNTs PEG-FITC Diameter 4 —9.21 sustained, with complete release reached after 20 hours. In Length 30 addition, the release kinetics significantly differed between Q-dots and PEG-FITC-SWNTs, with Q-dots being released 15 significantly faster than SWNTs. Loading Second Stage Nanoparticles into First Stage Silicon Control experiments performed with SP oxidized silicon Particles particles (FIG. 21, Panel f) and SP APTES modified silicon Protocols to efficiently load nanoparticles inside the pores particles (FIG. 21, Panel h) confirmed that Q-dots were not of first stage silicon particles were developed. One of the loaded inside the pores. Detachment of Q-dots from the factors affecting the loading process was the amount of 20 surfaces of SP silicon particles was massive and rapid second stage particles in the media surrounding the particles. (>80% in 15 min) suggesting that they were loosely inter- The analyzed fluorescent intensity of silicon particles loaded acting with the silicon particle surface. In contrast, the with increasing amounts of both Q-dots (FIG. 20, Panel A) kinetics of release of the PEG-FITC-SWNTs second stage and PEG-FITC-SWNTs (FIG. 20, Panel B) and demon- nanovectors from LP and SP silicon particles were compa- strated that particles loading was directly correlated with the 25 rable, confirming that PEG-FITC-SWNTs were loaded into second stage particles concentration. By raising the concen- the pores of first stage SP particles. tration of the second stage particles, progressively higher Confocal microscopy was used to further characterize levels of loading were achieved as assessed by flow cytom- loading of the second stage nanoparticles into the pores of etry (FIG. 20, Panels A and B) and by direct visualization the silicon carriers. FIG. 22, Panels a-b, show a series of 3 with fluorescent microscopy (FIG. 20, Panels C and D). 30 dimensional projections and rotations of LP APTES silicon These studies also demonstrated that surface chemical particles loaded with Carboxyl Q-dots. A more intense properties of both the first stage silicon particles and second fluorescence signal was detected in the central region of the stage particles affected loading efficiency and stability of the back face of the silicon particles, where larger pores are, see FIG. 19, Panel a. The less intense signal coming from the assembled multi-stage nanoparticulate carrier system. These 35 surrounding areas was due either to partial loading into observations were confirmed by the results shown in FIG. smaller pores or by the interaction of Q-dots with the surface 21, Panel a and Panel c, in which Carboxyl Q-dots, which of the silicon particle. had a negative surface charge (zeta potential —32.8 mV), and Multiple Loading PEG-FITC-SWNTs, with a negative surface charge (zeta Both red fluorescent Q-dots (emission 565 mu)and green potential —9.21 mV) could be more efficiently loaded into 40 fluorescent PEG-FITC-SWNTs, (emission 510 mu) were LP APTES modified silicon particles, than into the LP simultaneously loaded into the same nanoporous silicon oxidized silicon particles, which had a negative surface particles (FIG. 23A, Panels A and B). These studies dem- charge. Given the wide range of protocols available to onstrated that both types of second stage particles could be modify porous silicon',21-35,it is possible to efficiently load loaded into the same nanoporous silicon particle carriers. any kind of second stage particles by exploiting first and 45 The dynamics of multiple loading were different from the second stage surface chemistries. ones described for single loading and reached a stable Loading of second stage nanoparticles was a very rapid plateau after 60 minutes. The PEG-FITC-SWNTs were the process. FIG. 21, Panels a and c illustrate the results first to lodge inside the pores probably as a result of their obtained when LP oxidized silicon particles and LP APTES smaller size, while larger Q-dots took more time but reached modified silicon particles were incubated with a fixed 50 a higher level of loading (FIG. 23B, Panel Q. The release amount ofboth Amino and Carboxyl Q-dots and PEG-FITC- profiles of both types of second stage particles from the SWNTs. Complete loading of the first stage porous silicon silicon carrier remained almost unaltered (FIG. 23B, Panel particles occurred within 15 min if the combination of first D) compared to the observations of release, shown in FIG. stage porous silicon particles and second stage particles 21 Panel B. Confocal microscopy images shown in FIG. matched the electrostatic criteria described above. The load- 55 23C, Panels E-H, demonstrate that the two different second ing of SP oxidized silicon particles and SP APTES modified stage nanovectors preferentially localized to different areas silicon particles with second stage nanoparticles was also of the silicon particle. Given their larger size, Q-dots were evaluated (FIG. 21, Panels c and d, respectively). Q-dots exclusively found in the central area in association with size was between 13 and 16 mu so they could not be loaded larger pores. PEG-FITC-SWNTs were found around the into the pores of SP silicon particles (5-7 mu). On the 60 whole particle, with primary accumulation along the borders contrary, the pores of LP silicon particles (20-40 mu) were of the particle, in association with smaller pores. sufficiently large to allow Q-dots loading. As a consequence, When loaded silicon particles were incubated with SP particle mean fluorescence was only 6% of LP particles. HUVEC cells for 1 h at 37° C., second stage particles were Conversely, the smaller size of the PEG-FITC-SWNTs was released locally and selectively internalized by the cells that compatible with SP particle pores, resulting in increased 65 were reached by the first stage particles. Q-dots (FIG. 24, loading (25% of the PEG-FITC-SWNTs loaded into the LP Panels a-d) and PEG-FITC-SWNTs (FIG. 24, Panels e-h) silicon particles). entered the cytosol of the cells and resulted in significant US 10,143,658 B2 41 42 accumulation inside cytoplasmic vesicles. It was next affects stability ofthe assembled system and the time that the attempted to deliver both nanoparticles after having loaded second stage nanovectors remain loaded inside the nanop- them into the first stage silicon particle. Internalization of ores of the first stage silicon particles (FIG. 25, Panel B). both nanoparticles occurred with similar kinetics and Finally, a third mechanism that may be considered is the through the same mechanisms as suggested by the co- 5 electrostatic interaction between the first stage silicon par- localization of the fluorescent signals (FIG. 24, Panels j-m). ticle carrier and the second stage nanovectors (FIG. 25, In all cases, the 3.5 µm silicon particles on their own were Panel C). Taken together, each of these features may allow not internalized by the cells within the 1 h period. The bright the fine tuning of the amount of second stage nanovectors field images in FIG. 24 Panels d, h and m show the details that may be loaded into the first stage nanoporous silicon of particle morphology suggesting that they were associated 10 particles, which may be matched to a specific pharmaco- with the external cell surface rather than internalized by the logical need in future applications of drug delivery. cell, which was also verified by confocal imaging. The original and unique properties of the multi-stage Discussion nanocarrier system may allow multiple therapeutic agents, Silicon is widely used in the microelectronics industry, penetration enhancers or imaging contrast agents to be and has demonstrated its capacity for mass production of 15 delivered for the simultaneous detection and then destruc- microchips as well as for the development of biosensors, tion of tumors or for imaging and then treatment of other 36-39 implantable devices, and drug delivery systems While pathological conditions. bulk silicon remains inert in physiological buffer, porous silicon has a high degree of biodegradability with degrada- METHODS tion kinetics that may be finely tuned between hours to days 20 according to pore size density41,41 Porous silicon particles Fabrication of Porous Silicon Particles degrades to silicic acid, which is harmless to the body and A heavily doped p++ type Si(100) wafer with a resistivity we have found that neither whole intact silicon particles nor of 0.005 ohm cm-1 (Silicon their degradation products are cytotoxic for cells. The fab- Quest International, Santa Clara, Calif., USA)was used as rication protocols may allow the manufacturing of silicon 25 the substrate. A 200 mu layer of silicon nitride was deposited particulates of any desired shape, size (dimensions from 100 by low-pressure chemical vapor deposition (LPCVD) sys- nm to hundreds of µm), pore size (5-100 mu), and/or pore tem. Standard photolithography was used to pattern using a density. contact aligner(EVG 620 aligner). Silicon particles of small In addition, protocols to chemically modify the particles, diameter (100-500 mu) were obtained by flash imprint as attaching PEG to the surface of silicon particles in order 30 lithography. The nitride was then selectively removed by to reduce plasma protein adsorption, RES uptake, and to reactive ion etching (RIE). The photoresist was removed increases the circulation time of the particle as a drug with piranha (H2SO4:H2O2 3:1 (v/v)). The wafer was then delivery systems42 (manuscript in preparation) have been placed in a home-make Teflon cell for electrochemical developed. Successful conjugation of silicon particles to etching. The silicon particles with large pores (LP) were specific antibodies directed against ligands of therapeutic 35 formed in a mixture of hydrofluoric acid (49% HE) and importance such as vascular endothelial growth factor recep- ethanol (3:7 v/v) by applying a current density of 80 mA tor 2 (VEGFR2) and epidermal growth factor receptor CM-2 for 25 s. A high porosity layer was formed by applying (EGFR) and have evaluated their targeting and selective a current density of 320 mA cm2 for 6 s. For fabrication of cytotoxicity using an in-vitro cell culture model of primary silicon particles with small pores (SP), a solution of HE and HUVEC, as described above in Working Example 1. 40 ethanol was used with a ratio of 1:1 (v/v), with a current The present studies provide evidence of the ability of density applied of6 mAcrri2 for 1.75 min. The high porosity efficient loading of nanoporous silicon particles with second layers were formed by applying a current density of320 mA stage nanoparticles of different natures, sizes, and shapes. CM-2 for 6 s in an HF:ethanol mixture with a ratio of 2:5 The loading process may be regulated by adjustment of the (v/v). After removing the nitride layer by HF, particles were concentration of nanoparticles and/or by taking advantage of 45 released by ultrasound in isopropyl alcohol (IPA)for 1 min. the chemical surface properties of either or both first and The IPA solution containing porous silicon particles was second stage particles. The dynamics of nanoparticle release collected and stored at 4° C. and explored the ability of the nanoporous silicon particles BET Measurement to be loaded with multiple types of nanoparticles simulta- The nitrogen adsorption desorption volumetric isotherms neously has been shown in the present studies. These studies 50 obtained at 77 K were measured on a Quantachrome have clearly demonstrated that the Q-dots and PEG-FITC- Autosorb-3b BET Surface Analyzer. The samples were SWNTs used as second stage particles may localize to baked at 150° C. in vacuum overnight. Particle surface area different compartments of the first stage silicon nanoporous was obtained by BET linearization in the pressure range 0.05 particle carriers according to the size and chemical proper- to 1 P/P0. For the LP particles, the BET surface area was 156

ties of the second stage particles. Furthermore, the key 55 m2 g'- and pore volume was 0.54 cm3 g-1 using nitrogen mechanisms and the driving forces that are responsible for adsorption-desorption isotherm measurements. The average the loading and release processes have been identified and pore size was estimated to be about 24.2 mu. For SP ranked. The relative size of the pores of the silicon particles particles, the BET surface area was measured 294 m2 g-1; and of the second stage particles together may determine the pore volume was 0.38 cm3 g'- and the average pore size was most successful configuration of the combination of first and 60 7.4 mu. second stage particles (FIG. 25, Panel A). Concentration of Analysis of Particle Size and Concentration in Solution the second stage nanovectors influences the efficiency of the Particles were counted using a Z2 Coulter® Particle loading process, which may be regulated both by passive Counter and Size Analyzer (Beckman Coulter, Fullerton, diffusion and capillary convection of the second stage par- Calif., USA), with a 50 µm aperture size. The upper and ticles into the nanopores of the silicon particles. Hydrody- 65 lower size limits for analysis were set at 1.8 and 3.6 µm. For namic interactions of the second stage nanovectors with the analysis, particles were suspended in the balanced electro- nanopores of the silicon particles and Brownian motion lyte solution (ISOTON® II Diluent, Beckman Coulter Ful- US 10,143,658 B2 M, Ir! lerton, Calif., USA) and counted. The total volume of of the phthalimide moiety revealing the PEG-terminated original suspension of particles did not exceed 0.3% of the amine(H 2H-PEG-US-SWNT). Hydrazine monohydrate (10 final analysis volume. The particle counter displays the mL) was added to and the solution was heated to reflux concentration of particles in the solution as well an analysis under nitrogen for 12 h to give a terminal primary amine on of the particle size distribution. 5 the end of the PEGylated US-SWNT. The product was Oxidation of Silicon Microparticles purified by dialysis in DI water for 5 days. The solution was Silicon microparticles in IPA were dried in a glass beaker then transferred to a round bottom flask wrapped in foil and by heating (80-90° C.) and then oxidized them in a piranha FITC (0.12 g) predissolved in a small amount of DMF was solution (1:2 H202: concentrated H2SO4 (v/v)). The particles added to the H2H-PEG-US-SWNT solution. The reaction were added to a solution of H2O2 (30%), sonicated in a io was stirred at room temperature for 12 h. The solution was 551OR-MT ultrasonic cleaner(BRANSONIC, Danbury, CT, transferred to a dialysis bag and was kept in continuous USA)for 30 s and then H2SO4 (95-98%) was added and the dialysis with DI water in the dark for 5 days followed by suspension was heated to 100-110° C. for 2 h, with inter- filtration through glass wool to remove the undissolved mittent sonication to disperse the particles. The particles particulates. The final product FITC-PEG-US-SWNT con- were then washed with deionized (DI) water. Washing of the 15 tained some FITC physisorbed to the PEG-US-SWNTs particles in DI water involves centrifugation of the particu- instead of being covalently attached. Most of the phy- late suspension at 4,200 rpm for 5 min followed by removal sisorbed FITC still remains associated with the SWNTs after of the supernatant and resuspension of the particles in DI months of dialysis in water. water. The oxidized silicon particles were stored at 4° C. in Measurement of Zeta Potential of Silicon Particles, Q-dots, DI water until further use. 20 and SWNTs Surface Modification of Silicon Particles with 3-Aminopro- The zeta potential of the silicon particles, Q-dots, and pyltriethoxysilane (APTES) FITC-PEG-US-SWNTs were analyzed using a Zetasizer Prior to silanization, the oxidized silicon particles were nano ZS (Malvern Instruments Ltd., Southborough, Mass., hydroxylated in 1.5 M HNO3 for approximately 1.5 h. The USA). The particles were suspended in 20 mM Tris(hy- particles were then washed 3-5 times with DI water followed 25 droxymethyl)aminomethane (TRIS-HCL) buffer (pH 7.3) by 2 washes with IPA. The silicon particles were then for the analysis. suspended in IPA containing 0.5% (v/v) APTES for 45 min Loading of Q-dots and SWNTs Into First Stage Silicon at room temperature. The particles were then washed with Particles IPA (4,200 g for 5 min, 5 times), and stored in IPA at 4° C. The silicon nanoporous particles that were used in devel- Q-dots and Water Fluorescently Labeled Water-Soluble 30 opment of the multi-stage nanodevice include LP oxidized SWNTs. porous silicon, SP oxidized porous silicon and APTES Q-dots were purchased from Invitrogen (Carlsbad, Calif., modified LP and SP particles. The second stage particles USA). In this study, we used Amino-PEG with 525 nm and include Amino-PEG Q-dots, Carboxyl Q-dots, and PEG- 565 nm emission wavelength (catalogue number FITC-SWNTs. Initial experiments were performed to titrate Q2154IMP and Q2153IMP respectively) and Carboxyl 35 the loading of the second stage particles into the first stage Q-dots with 525 nm and 565 nm emission wavelength silicon particles. For each experimental point of the titration (catalogue number Q2134IMP and Q2133IMP respec- experiments, 3.0x105 silicon particles were used, which tively). To produce ultra-short (US)-SWNTs 43 that are were resuspended in low binding polypropylene micro cen- heavily sidewall carboxylated, oleum (20% free SO3, 25 trifuge tubes(VWR International, West Chester, Pa., USA) mL) was added to purified HiPco SWNTs (0.100 g) under 40 containing 3µl DI water. For every experiment, the given nitrogen in round bottom flask and the mixture was stirred molarity of TRIS-HCl solution was adjusted at pH 7.3. overnight to disperse the SWNTs (HiPco SWNTs were Either 2µM Q-dots (5 µl) or 20 ng/µl PEG-FITC-SWNTs (9 obtained from the Rice University HiPco laboratory. For the µl) were added to the TRIS-HCl solution, with 20 µl as the purification established procedures were followed.44 In a final volume for the loading experiments. Samples were separate flask, oleum (25 mL, 20% free SO3)was slowly 45 incubated by being placed on a rotating wheel (20 rpm)for added to concentrated nitric acid (18 mL). This mixture was 15 min at 25° C. After incubation, the samples were diluted immediately added to the SWNTs and the SWNTs were with 20 mM TRIS-HCl, pH 7.3 to a volume of 150 µl and heated to 60° C.for 2 h. The solution was then slowly poured promptly examined for fluorescence intensity using a FAC- over ice and filtered over a 0.22 µm polycarbonate mem- Scalibur (Becton Dickinson)flow cytometer. To evaluate the brane. The filter cake was thoroughly washed with water. 50 concentration dependent loading of Q-dots and PEG-FITC- The vacuum was removed and the cake was dissolved in a SWNTs into silicon particles, we used 3.0x105 silicon minimal amount of methanol after which ether was added particles and an incubation time of 15 min, with either 0.01, leading to flocculation of the US-SWNTs and the vacuum 0.1, 1, 10, 100, 1,000 and 2,000 nM Q-dots or 0.05, 0,1, 2.5, was reapplied. Ether was continually added until a neutral 10 and 20 ng/µl ofPEG-FITC-SWNTs. To evaluate the time pH was achieved from the washings. The US-SWNT cake 55 dependent loading, 3.0x105 first stage particles, 2,000 nM was dried under vacuum. To obtain PEGylated SWNTs, Q-dots or 20 ng/µl of PEG-FITC-SWNTs with incubation US-SWNTs (0.034 g, 3.1 meq C) were dispersed in dry times of 15, 30, 45 and 60 minutes were used. For evaluation DMF (30 mL) using sonication (bath or cup-horn, and of loading of silicon particles with both Q-dots and PEG- Model) for 30 min in a round bottom flask. To this, Dicy- FITC-SWNTs, 3.0x105 silicon particles, 1,000 nM Q-dots, clohexylcarbodiimide(DCC) (0.32 g, 1.5 mmol), and 5,200 6o and 10 ng/µl ofPEG-FITC-SWNTs were used with the same MW of hydroxyethyl phthalimide terminated PEG (0.32 g) final volume of incubation. were added and the mixture stirred under nitrogen for 24 h. Studies of release of Q-dots and SWNTs from first stage The solution was transferred to a dialysis bag (CelluSep Hl, silicon particles. 50,000 MWCO, Part #: 1-5050-34, Membrane Filtration LP silicon particles (2.1 x 106), which were either oxidized Products)for 5 d and the product filtered through glass wool 65 or APTES modified, were combined with 2µM Amino-PEG to remove any particulate that formed in the dialysis bag. Q-dots or Carboxyl Q-dots in a 200 mM TRIS-HCl solution The contents of the dialysis bag were subjected to removal at pH 7.3 or with 20 ng/µl PEG-FITC-SWNTs in a 20 mM US 10,143,658 B2 45 46 TRIS-HC1 solution at pH 7.3 or with both 1µM Q-dots and Bright Field Microscopy 10 ng/µl PEG-FITC-SWNTs in a 50 mM TRIS-HCl solution Particles were analyzed in bright field contrast with an at pH 7.3. The final incubation volume for all studies was Olympus CKX41 microscope with a 40x magnification lens. 140 µl. Samples of first stage silicon particle carriers and Images were taken with an SP-350 Olympus True-Pic second stage particles were incubated using a rotating wheel 5 TURBO Image Processor camera. (20 rpm) for 15 min at 25° C. The solution containing the Fluorescent Microscopy first stage silicon particles and second stage particles were Fluorescent imaging of particles was performed with a then washed in 1.4 mL DI H2O, and then centrifuged for 5 Nikon Eclipse TE2000-E with a DQC-FS Nikon CCD min at 4,200 rpm in a Beckman Coulter Allegra X-22 Camera kept at —30.1' C. All the samples were mounted centrifuge. Pellets present after centrifugation were then 10 immediately before the analysis and the images acquired resuspended in 70 µl of DI H2O and 10µl was removed from with a 63x immersion oil objective. The microscope settings each vial to assess the fluorescence ofthe samples using flow were kept constant throughout all the experiments. numeri- cytometry. Fluorescence was recorded at time 0 and then cal aperture was set at 1.4, refractive index at 1.515, expo- over 6 time points, which included 30, 60, 90, 180, 360, and 15 sure time at 500 ms, readout Speed at 10 MHz and conver- 1,200 min. The residual 60 µl left in each vial was diluted to sion gain at 1/6x. 3 ml in 20 mM TRIS-HCl 0.9% NaCl release buffer, The images were analyzed and measured with the NIS followed by incubation using a rotating wheel (20 rpm) for Elements AR 2.3 Software. the given amount of time (30, 60, 90, 180, 360 and 1,200 Confocal Microscopy min) at 37° C. At each time point, samples were centrifuged 20 Confocal imaging of particles was performed with a for 5 min at 4,200 rpm and the fluorescence evaluated using LEICA DM6000 microscope. All the samples were mounted flow cytometry. immediately before the analysis and the images acquired Flow Cytometry Setup with a HCX PL APO CS 63x immersion oil objective with Particles were assessed for fluorescence using a FAC- a 1.4 numerical aperture and 1.52 refraction index. For all Scalibur (Becton Dickinson). Bivariate dot-plots defining 25 the acquisitions, the pinhole was set at 95.6µm (1 Airy unit), logarithmic side scatter (SSC) versus logarithmic forward both 488 nm argon and 561 mn lasers were at 15% of their scatter(FSC) were used to evaluate the size and shape of the capacity; the scan speed was set at 400 Hz. The single green silicon particles (3 µm in diameter, 1.5 µm in height) and to fluorescence imaging photomultiplier voltage was set at 750 exclude non-specific events from the analysis. Control rain- V. For dual color imaging, the PMT for the red channel was bow BD CalibriteTM beads (3.5 µm in size) were used to 30 set at 600 V while the PMT for the green channel was set at calibrate the instrument. A polygonal region (RI) was 1000 V. To improve the image quality, 2-frame accumula- defined as an electronic gate around the centre of the major tion, 2-line accumulations and 4-frame averages were per- population of interest, which excluded events that were too formed during acquisitions. Final voxel width and height close to the signal-to-noise ratio limits of the cytometer. For were 19.8 mu. The images were magnified digitally (10x) each sample, the number of particles detected within the RI 35 and a median adjustment (3 pixels, 2 rounds) was used region was above 90%. For analysis of the geometric mean during the post-processing using the LAS AF 1.6.2 software. fluorescence intensity(GMFI), dot-plots were created which compared fluorescence channel 1 (FLI) and FL2 versus log WORKING EXAMPLE 3 FSC with analysis of events falling within the gated region defined as RL The peaks identified in each of the samples 4o Liposomes in 3.5 Micron Silicon Particles were analyzed in the corresponding fluorescent histogram Fluorescently labeled siRNA loaded 1,2-dioleoyl-sn- and the geometric mean values recorded. For particle detec- glycero-3-phosphaticholine (DOPC) liposomes were pre- tion, the detectors used were FSC E-1 and SSC with a pared as detailed in C. N. Landen Jr., et al. Cancer Res. 2005, voltage setting of 474 volts (V). The fluorescent detector 65(15), 6910-6918, and J. Clin. Cancer Res. 2006; 12(16), FLl was set at 800 V. Green fluorescence(FITC and Q-dots 45 4916-4924. 525) was detected with FL1 using a 530/30 nm band-pass Fluorescently labeled siRNA loaded DOPC liposomes filter. Red fluorescence (Q-dots 565) was detected using (original siRNA concentration: 100 ng/µl), as second stage FL2. When single color detection only was analyzed, color particles, were mixed with 1 st stage large pore "LP" oxi- compensation was set at zero, and when dual red-green color dized silicon particles (3.5 micron). Incubation was per- detection was performed, FL compensation was set at 25% 50 formed in 20 mM Tris pH 7.3 for 30 min at room tempera- of FL2 and FL2 compensation was set at 35% of FLl. ture. The solution was spun down at 4,200 rpm for 1 min at Instrument calibration was carried out before, in between, room temperature. The supernatant was recovered and the and after each series of experiments for data acquisition fluorescence of the sample was measured by fluorimetry using BD CalibriteTM beads. using 544 nm/590 mn (excitation/emission) settings. The Scanning Electron Microscopy 55 particle pellet comprising the 1 st stage particles that had The morphology of the silica particles was acquired using incorporated the fluorescent liposomes were resuspended in scanning electron microscopy (SEM, model LEO 1530). Par- 100 µl of deionized water and fluorimetric readings were ticles were directly placed on SEM samples stage and dried. taken. The loading time dynamics of Fluorescently labeled For the particles tested in the high salt buffers, a mild siRNA loaded DOPC liposomes into 3.5 micron LP and SP washing step in DI water was performed before putting on 60 particles is shown in FIG. 813, Panel D. In FIG. 813, Panels the stage. The acceleration voltage was 10 kV. E and F show fluorescent microscopy images visualizing Tapping Mode Atomic Force Microscopy (AFM) second stage liposomes containing Alexa 555 labeled AFM samples were prepared by deposition from DMF SiRNA into 3.5 micron nanoporous silicon first stage par- onto a freshly cleaved mica surface. Samples were spin ticles LP in white field and red field respectively coated and sectional analysis was used to determine the 65 Alexa 555 fluorescently labeled siRNAs were encapsu- heights of each sample. AFM samples were obtained using lated into liposomes and loaded into the 1st stage porous tapping mode. silicon particles. The data in FIG. 26, Panel A, show that the US 10,143,658 B2 47 48 fluorescence associated with the porous Silicon carrier tion may be determined by lysis with methanol(30% of final increased with the amount of nanoliposomes. volume) and measuring the UV absorbance at 480 nm. To test the release ofliposome from 1 st stage particles, the assembled multistage systems were incubated with 10% REFERENCES fetal bovine serum (pH 7.4) and release of nanoliposomes from the 1 st stage particles was followed along time using The following references are cited in the foregoing text: fluorimetry. Complete unloading was achieved in about 36 1. Cheng, M. M. et al. Nanotechnologies for biomolecular h, see FIG. 26, Panel B. detection and medical diagnostics. Curr Opin Chem Biol 10, 11-9 (2006). WORKING EXAMPLE 4 10 2. Ferrari, M. Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer 5, 161-71 (2005). Liposomes to Silicon Particles 3. Moghimi, S. M., Hunter, A. C. & Murray, J. C. Nano- Preparation of Liposomes: medicine: current status and future prospects. Faseb J 19, Liposomes with a lipid composition of 58:40:2 (Mol %) 311-30 (2005). DPPC: Cholesterol: DSPE-Methoxy PEG (2000) respec- 15 tively may be made by the extrusion process as follows: 4. Sullivan, D. C. & Ferrari, M. Nanotechnology and tumor Briefly, the lipids may be dissolved in ethanol at 55 oC. The imaging: seizing an opportunity. Mol Imaging 3, 364-9 dissolved lipids may then be hydrated with 300 mM ammo- (2004). nium sulfate solution (for 15-30 minutes) to facilitate active 5. Wang, M. D., Shin, D. M., Simons, J. W. & Nie, S. loading of doxorubicin, see Li, et al. Biochim et Biophys 20 Nanotechnology for targeted cancer therapy. Expert Rev Acta 1415 (1998). Liposomes may be extruded through a Anticancer Ther 7, 833-7 (2007). series of Nuclepore track-etched polycarbonate membranes 6. Lasic, D. D. Doxorubicin in sterically stabilized lipo- of decreasing pore sizes. The liposomes may then be somes. Nature 380, 561-2 (1996). extruded 5 times through a 0.2 µm membrane. This may be 7. Uziely, B. et al. 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Current Opinion in Chemical Biology 2005, the delivery of therapeutics. Expert Opin Drug Deliv 4, 9:343-346. 101-10 (2007). 75. Ferrari M., Nat. Rev. Cancer. 2005 5(3):161-71. 41. Canham, L. T. Bioactive silicon structure fabrication 60 Although the foregoing refers to particular preferred through nanoetching techniques. Advanced Materials 7, embodiments, it will be understood that the present inven- 1033-& (1995). tion is not so limited. It will occur to those of ordinary skill 42. Zhang, M., Desai, T. & Ferrari, M. Proteins and cells on in the art in light of this disclosure that various modifications PEG immobilized silicon surfaces. Biomaterials 19, 953- may be made to the disclosed embodiments and that such 60 (1998). 65 modifications are intended to be within the scope of the 43. Chen, Z. et al. Soluble ultra-short single-walled carbon present invention. All ofthe publications, patent applications nanotubes. JAm Chem Soc 128, 10568-71 (2006). and patents cited in this specification are incorporated herein US 10,143,658 B2 51 52 by reference to the extent that they provide details and a basement membrane permeation enhancer, a tight junction description consistent with and supplemental to this disclo- protein (tjp) permeation enhancer and any combination sure. thereof. The invention claimed is: 10. The composition of claim 1, wherein the first stage 1. A multistage delivery composition comprising: 5 particle is configured to release the second stage particle in a first stage particle containing second stage particle, response to an external stimulus. wherein the first stage particle, comprises: 11. A method comprising administering to a subject a (i) a body; composition comprising: (ii) at least one surface with a surface charge; and a first stage particle containing a second stage particle, (iii) a plurality of nanoscale reservoirs in the body of the to wherein the first stage particle comprises: first stage particle, (i) a body; wherein each reservoir is connected to at least one surface (ii) at least one surface with a surface charge; and of the body through at least one channel, wherein the at least one channel is in the form of a pore (iii) a plurality of nanoscale reservoirs in the body of the open to the surface, 15 first stage particle, wherein at least one of the plurality of the nanoscale wherein each reservoir is connected to at least one surface reservoirs contains the second stage particle in the pore of the body through at least one channel, open to the surface, and wherein: wherein the at least one channel is in the form of a pore the first stage particle is biodegradable and has a selected open to the surface, non-spherical shape; 20 wherein at least one of the plurality of the nanoscale the first stage particle is a porous micro or nanoparticle reservoirs contains the second stage particle in the pore with a size from about 100 mn to about 5 microns; open to the surface, and wherein: the second stage particle encapsulates a therapeutic agent; the first stage particle is biodegradable and has a selected the first stage particle is configured to overcome a first non-spherical shape; biological barrier; and 25 the first stage particle is a porous micro or nanoparticle the second stage particle is configured to overcome a with a size from about 100 mn to about 5 microns; second biological barrier, wherein the first stage par- the second stage particle encapsulates a therapeutic agent; ticle and the second stage particle are configured to the first stage particle is configured to overcome a first sequentially overcome the first biological barrier and biological barrier; and the second stage particle is second biological barrier respectively. so configured to overcome a second biological barrier, 2. The multistage delivery composition of claim 1 wherein the first stage particle and the second stage wherein the first stage particle is configured to release the particle are configured to sequentially overcome the second stage particle for delivery of the therapeutic agent. first biological barrier and second biological barrier 3. The multistage delivery composition of claim 1 respectively. wherein the first stage particle is configured to release the 35 12. The method of claim 11 wherein the first stage particle second stage particle for delivery of the therapeutic agent via is configured to release the second stage particle for delivery diffusion ofthe second stage particle through the channels in of the therapeutic agent. the first stage particle. 13. The method of claim 11 wherein the first biological 4. The multistage delivery composition of claim 1 barrier and the second biological barrier are each indepen- wherein the first stage particle is configured to release the 4o dently selected from the group of biological barriers con- second stage particle for delivery of the therapeutic agent via sisting of a hemo-rheology barrier, a reticulo-endothelial degradation or erosion of the body the first stage particle. system barrier, an endothelial barrier, a blood brain barrier, 5. The multistage delivery composition of claim 1 a tumor-associated osmotic interstitial pressure barrier, an wherein the first biological barrier and the second biological ionic and molecular pump barrier, a cell membrane barrier, barrier are each independently selected from the group of 45 an enzymatic degradation barrier, a nuclear membrane bar- biological barriers consisting of a hemo-rheology barrier, a rier, and any combination of thereof. reticulo-endothelial system barrier, an endothelial barrier, a 14. The method of claim 11 wherein the first stage particle blood brain barrier, a tumor-associated osmotic interstitial comprises a chemical targeting moiety disposed on the pressure barrier, an ionic and molecular pump barrier, a cell surface of the first stage particle, wherein said chemical membrane barrier, an enzymatic degradation barrier, a 50 targeting moiety comprises at least one moiety selected from nuclear membrane barrier, and any combination of thereof. the group consisting of dendrimer, an aptamer, an antibody, 6. The multistage delivery composition of claim 1 a biomolecule and any combination thereof. wherein the first stage particle comprises a chemical target- 15. The method of claim 11 wherein the at least one of the ing moiety disposed on the surface of the first stage particle, plurality of nanoscale reservoirs contains at least one addi- wherein said chemical targeting moiety comprises at least 55 tional agent. one moiety selected from the group consisting of dendrimer, 16. The method of claim 11 wherein the at least one an aptamer, an antibody, a biomolecule and any combination additional agent comprises at least one penetration enhancer, thereof. at least one additional active agent, or at least one targeting 7. The multistage delivery composition of claim 1 moiety. wherein at least one of the plurality of nanoscale reservoirs 60 17. The method of claim 11 wherein the composition is contains at least one additional agent. administered intravascularly. 8. The composition of claim 7, wherein the at least one 18. A method of fabricating a multistage delivery com- additional agent comprises at least one penetration enhancer, position, comprising: at least one additional active agent, or at least one targeting (a) providing a first stage particle, wherein the first stage moiety. 65 particle comprises: 9. The composition of claim 8, wherein the at least one (i) a body; penetration enhancer is selected from the group consisting of (ii) at least one surface with a surface charge; and US 10,143,658 B2 53 54 (iii) a plurality of nanoscale reservoirs in the body of the 22. The of method claim 18, wherein loading the second first stage particle, stage particle inside one of the plurality of nanoscale reser- wherein each reservoir is connected to at least one voirs of the first stage particle comprises loading via passive surface of the body through at least one channel, diffusion or capillary convection, or by a combination of wherein the at least one channel is in the form of a pore 5 those. open to the surface, and wherein: 23. The method of claim 18 wherein loading the second the first stage particle is biodegradable and has a selected stage particle inside one of the plurality of nanoscale reser- non-spherical shape; voirs of the first stage particle comprises modifying a the first stage particle is a porous micro or nanoparticle surface charge of the first particle or the second particle. with a size from about 100 mn to about 5 microns; 10 24. The multistage delivery composition of claim 1, (b) providing a second stage particle; wherein the second stage particle comprises a plurality of and second stage particles stacked within the pore open to the (c) loading the second stage particle inside one of the surface. plurality of nanoscale reservoirs of the first stage par- 25. The method of claim 11, wherein the second stage ticle such that the nanoscale reservoir contains the 15 particle comprises a plurality of second stage particles second stage particle in the pore open to the surface, stacked within the pore open to the surface. and such that the first stage particle is configured to 26. The method of claim 18, wherein the second stage overcome a first biological barrier and the second stage particle comprises a plurality of second stage particles particle is configured to overcome a second biological stacked within the pore open to the surface. barrier, wherein the first stage particle and the second 20 27. The multistage delivery composition of claim 1, stage particle are configured to sequentially overcome wherein the second stage particle comprises at least one the first biological barrier and second biological barrier constituent selected from the group consisting of a liposome, respectively. a micelle, an ethosome, a carbon nanotube, a fullerene 19. The method of claim 18, wherein loading the second nanoparticle, a metal nanoparticle, a semiconductor nano- stage particle inside one of the plurality of nanoscale reser- 25 particle, a polymer nanoparticle, an oxide nanoparticle, a voirs of the first stage particle comprises placing the first viral particle, a polyionic particle and a ceramic particle. stage particle and the second stage particle in a sealed 28. The method of claim 11, wherein the second stage chamber having a pressure above atmospheric pressure. particle comprises at least one constituent selected from the 20. The method of claim 19 wherein first stage particle group consisting of a liposome, a micelle, an ethosome, a and the second stage particle are submerged in a solution in 30 carbon nanotube, a fullerene nanoparticle, a metal nanopar- the sealed chamber. ticle, a semiconductor nanoparticle, a polymer nanoparticle, 21. The method of claim 20 wherein the first stage particle an oxide nanoparticle, a viral particle, a polyionic particle is subjected to reduced pressure prior to being placed in the and a ceramic particle. solution in the sealed chamber.